JP2020525445A - Cation exchange chromatography wash buffer - Google Patents

Abstract

A wash buffer containing a detergent for use in affinity and cation exchange chromatography purifies the protein of interest from protein aggregates, removes the virus, and / or inactivates it. When used during affinity or cation exchange chromatography for purification of proteins of interest, such as antibodies, wash buffers significantly improve virus elimination from this preparation while host cell-derived proteins. And also reduce the level of protein aggregates. After affinity or cation exchange chromatography with wash buffer, the protein of interest can be further purified using other chromatography and filtration procedures.

Description

BACKGROUND OF THE INVENTION The present invention relates generally to the field of protein biochemistry. More particularly, the invention relates to wash buffers used in cation exchange chromatography to purify target proteins, eg, antibodies, from protein aggregates, to remove and/or inactivate viruses. It has been found that washing the cation exchange chromatography support with a buffer containing one or more detergents substantially improves viral clearance from the protein preparation.

Recombinant proteins are expressed in host cells and are typically purified by a series of chromatography and filtration steps. Traditionally, a monoclonal antibody (mAb) purification process flow consists of three chromatography columns. The main functions of each chromatography column are as follows.
Protein A (ProA) for product recovery and host cell derived protein (HCP) removal;
Cation exchange (CEX) for removal of DNA, HCP and aggregates; and anion exchange (AEX) for further DNA and HCP removal and virus clearance.
The general purification scheme, also known as the downstream cascade, uses affinity chromatography followed by cation exchange chromatography (CEX) or hydrophobic interaction chromatography (HIC) followed by anion. With exchange chromatography (AEX). CEX chromatography and AEX are commonly regarded as polishing steps (eg, GE Healthcare. (2006). Process-scale purification of monoclonal antibodies-polishing esginge gels gingesge/geesge/geesge/geesge. %20Content/Files/1314750913712/litdoc28903716_20130507212449.PDF). In addition, typical downstream purification schemes also include a virus inactivation step and a virus filtration step to ensure proper virus clearance.

Unfortunately, each additional chromatography step generally reduces overall protein yield. Moreover, the use of additional chromatographic steps adds to the cost and complexity of operating the process.

Reducing or eliminating the polishing chromatography step by improving the performance of the cation exchange chromatography step significantly reduces development effort and manufacturing costs, improves target product recovery and improves the manufacturing process. It is possible to simplify. However, one of the main concerns of using CEX as the only polishing step is that it is not considered a robust virus clearance step (as shown by metadata analysis from multiple studies. Miesegaes et al. Biotechnol. Bioeng. 2010; 106:238-246).

The operational design space is significantly limited in terms of process parameters for CEX steps to achieve adequate process performance, and especially effective virus clearance (Miesegaes, GR et al. Biotechnol. Bioeng. 109:2048-20582, and Connell-Crowley, L. et al. Biotechnol. Bioeng. 2012; 109:157-165).

GE Healthcare. (2006) Miesegaes et al. Biotechnol. Bioeng. 2010; 106:238-246. Miesegaes, G.; R. et al. Biotechnol. Bioeng. 2012;109:2048-20582. Connell-Crowley, L.; et al. Biotechnol. Bioeng. 2012;109:157-165.

The present invention provides a wash procedure applied to the cation exchange step to improve chromatographic performance, and in particular viral clearance of this step. The application of a wash procedure, and its associated robust viral clearance capacity, allows for the removal of certain other polishing chromatography steps, such as AEX, and can reduce the number of chromatography steps required. ..

Also, application of the wash procedure described herein can allow removal of the virus inactivation step, which may occur under common virus inactivation conditions (eg, low pH) or virus inactivation. Particularly useful for unstable molecules in processes where activation may be difficult to perform (eg, in continuous processing) and yet is suitable for chromatographic downstream unit operations and virus filtration steps. Elimination can be achieved.

It has been found that by improving the virus clearance capacity on the CEX support, it is possible to eliminate the AEX chromatography step and simplify the entire manufacturing process for proteins such as antibodies. Addition of detergent to the wash buffer in CEX improves virus clearance without compromising impurity clearance. This improvement in the virus exclusion capacity of the CEX column is utilized either as the CEX is utilized as a polishing step (ie after the primary product recovery or capture step, eg on ProA etc.) or as the primary recovery step itself. It is observed regardless of whether or not.

The wash solution can be used in a protein purification method, such as a multi-step protein purification method as described above. In another aspect, the disclosure features a method of purifying a protein of interest expressed from a cell, including by recombinant expression or by hybridoma expression. Generally, this method involves loading a mixture of a protein of interest and one or more contaminating viruses on a CEX chromatography support and washing the support with an aqueous wash solution containing a detergent prior to application. Eluting the protein from the support to form a purified eluate of the protein of interest, the purified eluate being free of contaminating virus (or loaded onto a CEX chromatography support). Including its reduced number compared to the mixture). Optionally, the method comprises washing the support with a detergent-free intermediate wash buffer after the detergent wash step and before the elution step. This intermediate wash buffer removes any residual detergent from the previous wash from the support prior to the elution step.

The mixture of proteins of interest can be the eluent of the previous chromatography step, such as the eluent of an affinity chromatography column, optionally a protein A chromatography column. In another embodiment, the mixture of proteins of interest is harvested cell culture fluid (HCCF). In this embodiment, the protein purification method may not include an affinity chromatography step (ie, the CEX chromatography step described herein may replace the affinity chromatography, eg, Protein A step). it can). The aqueous wash liquor can be any such solution described or illustrated herein, including those wash fluids described in the sections below. The intermediate wash buffer is any aqueous CEX wash buffer, such as a CEX equilibration buffer, or any of the CEX wash solutions described herein under detergent-free conditions. You can In one embodiment, the intermediate wash buffer is of the same composition as any one of the CEX wash solutions described herein, without detergent.

In addition, the method comprises acidifying the purified eluate containing the protein of interest (eg, reducing the pH of the eluate) to inactivate any residual virus in the eluate. You can After lowering the pH for a period of time sufficient to inactivate the virus in the eluate, the pH rises to a more neutral pH. Further, the method can include filtering the purified eluate of the protein of interest to remove virus, including inactivated virus. Further, the method can include treating the purified eluate of the protein of interest by diafiltration, ultrafiltration, or both diafiltration and ultrafiltration. The method can further include formulating the purified eluate of the protein of interest as a composition that includes a pharmaceutically acceptable excipient. The method can further include formulating the further purified eluate of the protein of interest as a composition comprising a pharmaceutically acceptable excipient. In some embodiments, the protein purification method does not include an anion exchange (column or membrane) chromatography step. In some embodiments, the protein purification method does not include a separate polishing chromatography step. In some embodiments, the protein purification method does not include a virus inactivation step.

In some embodiments, a method of purifying a protein of interest bound to a cation exchange (CEX) chromatographic support comprises applying a wash solution containing one or more detergents to the CEX chromatographic support. Including. In some embodiments, the method further comprises eluting the protein of interest from the support to form a purified eluate containing the protein of interest.

In some embodiments, a method of purifying a protein of interest comprises loading on a CEX chromatography support a mixture comprising the protein of interest and one or more contaminating proteins, aggregates, and/or viruses, Washing the support with a wash solution containing one or more detergents and then eluting the protein of interest from the support to form a purified eluate of the protein of interest.

In some embodiments, a method of purifying a protein of interest comprises loading on a CEX chromatography support a mixture comprising the protein of interest and one or more contaminating proteins, aggregates, and/or viruses, After washing the support with an aqueous wash containing one or more surfactants to elute one or more contaminating proteins, aggregates, and/or viruses from the support, the protein of interest is removed from the support. Eluting to form a purified eluate of the protein of interest.

In some embodiments, the one or more surfactants are Triton 100, nonylphenoxypolyethoxylethanol (NP40), sulfobetaine-12 (SB-12), sulfobetaine-14 (SB-14), lauryldimethylamine N. -Oxide (LDAO), polysorbate 20 (PS20), polysorbate 80 (PS80), or combinations thereof. In some embodiments, the one or more surfactant comprises a zwitterionic surfactant. In some embodiments, the zwitterionic surfactant is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), or cocamidopropyl hydroxysultaine (CAHS), or Is a combination of. In some embodiments, the one or more surfactant comprises a quaternary ammonium salt. In some embodiments, the quaternary ammonium salt is cetrimonium bromide (CTAB) or dioctadecyldimethylammonium bromide (DODAB). In some embodiments, the one or more surfactant comprises an alkylphenol ethoxylate. In some embodiments, the alkylphenol ethoxylate is nonoxynol, optionally nonaethylene glycol. In some embodiments, the one or more surfactant comprises ethoxylate. In some embodiments, the one or more surfactant comprises an alkyl polyglucoside, optionally decyl glucoside. In some embodiments, the one or more surfactant comprises an amino oxide. In some embodiments, the one or more surfactant comprises phosphine oxide. In some embodiments, the one or more surfactant comprises a nonionic surfactant. In some embodiments, the one or more surfactant comprises an anionic surfactant, optionally the anionic surfactant is sodium dodecyl sulfate (SDS).

In some embodiments, the wash liquor comprises about 0.01 w/v% to about 1 w/v% of one or more surfactants. In some embodiments, the wash liquor comprises from about 0.05 w/v% to about 0.5 w/v% of one or more surfactants. In some embodiments, the wash liquor comprises about 0.1 w/v% of one or more surfactants. In some embodiments, the wash liquor comprises about 0.2 w/v% of one or more surfactants. In some embodiments, the wash liquor comprises about 0.5 w/v% of one or more surfactants.

In some embodiments, the CEX chromatography support is Sepharose matrix resin. In some embodiments, the CEX chromatography support is a synthetic polymer matrix resin. In some embodiments, the CEX chromatography support is not a hydrophilic resin. In some embodiments, the CEX chromatographic support is Capto S, Fractogel EMD SO3-, Fractogel EMD TMAE, Fractogel EMD, Eshnumo S, Eshmuno HCp, Tohyo Toyopolar SP, Tosoh Toyopearl, Toh, Toyopoyl SP, Toh, Toyopoyl SP, Toh, Toyopearl, Toh. To be done.

In some embodiments, the method further comprises applying a subsequent wash solution to the CEX chromatography support prior to eluting the protein of interest from the support. In some embodiments, this subsequent wash solution is free of surfactant. In some embodiments, this subsequent wash removes surfactant from the support. In some embodiments, applying this subsequent wash increases the removal of host cell-derived protein (HCP) compared to not applying this subsequent wash.

In some embodiments, the method further comprises loading harvested cell culture fluid (HCCF) onto the support prior to applying a wash fluid containing one or more detergents to the support. In some embodiments, the mixture comprising the protein of interest and one or more contaminating proteins, aggregates, and/or viruses is HCCF.

In some embodiments, the method does not include Protein A purification.

In some embodiments, the method further comprises loading the eluate of the chromatography step onto the support prior to applying the wash solution containing one or more detergents to the support. In some embodiments, the mixture containing the protein of interest and one or more contaminating proteins, aggregates, and/or viruses is the eluate of a chromatography step. In some embodiments, the eluent of the chromatography step is the eluent of the affinity chromatography step. In some embodiments, the affinity chromatography step is Protein A purification. In some embodiments, Protein A purification comprises washing the Protein A support with a wash solution containing one or more detergents. In some embodiments, Protein A purification does not include washing the Protein A support with a wash solution containing one or more detergents.

In some embodiments, the wash solution excludes virus as measured by LRV.

In some embodiments, the protein purification method comprises: a) loading onto a Protein A chromatography support a mixture comprising the protein of interest and one or more contaminants, aggregates, and/or viruses, b. B.) washing the support with a Protein A chromatography wash containing one or more detergents to remove one or more contaminating proteins, aggregates and/or viruses from the Protein A chromatography support, c. E) eluting the protein of interest from the protein A chromatography support, d) loading an eluate containing the protein of interest on a cation exchange (CEX) chromatography support, e) one or more interfaces. Washing the CEX chromatography support with a CEX chromatography wash containing an activator to remove one or more contaminating proteins, aggregates and/or viruses from the support, and f) CEX chromatography the protein of interest. Eluting from the chromatographic support to form a purified eluate of the protein of interest.

In some embodiments, the contaminating protein is a host cell derived protein. In some embodiments, the host cell derived protein is derived from Chinese Hamster Ovary cells.

In some embodiments, the Protein A wash eliminates virus as measured by log reduction value (LRV). In some embodiments, the CEX lavage excludes virus as measured by LRV.

In some embodiments, the LRV is between about 1.0 and about 10.0 log ₁₀ . In some embodiments, the LRV is between about 1.0 and about 5.0 log ₁₀ . In some embodiments, the LRV is between about 4.0 and about 8.0 log ₁₀ . In some aspects, the LRV is about 3.0 log ₁₀ . In some aspects, the LRV is about 4.0 log ₁₀ . In some aspects, the LRV is about 5.0 log ₁₀ . In some aspects, the LRV is about 6.0 log ₁₀ .

In some embodiments, the LRV is measured by quantitative PCR. In some embodiments, the LRV is measured using an infection assay.

In some embodiments, this LRV is achieved by applying one column volume (CV) wash. In some embodiments, this LRV is achieved by applying a wash of 2CV. In some embodiments, this LRV is achieved by applying a wash of 3CV. In some embodiments, this LRV is achieved by applying a wash of 4CV. In some embodiments, this LRV is achieved by applying a 5 CV wash.

In some embodiments, the method further comprises applying a detergent-free wash solution to the support prior to applying the wash solution containing one or more surfactants to the support.

In some embodiments, the method further comprises treating the purified eluate of the protein of interest by diafiltration, ultrafiltration, or both diafiltration and ultrafiltration.

In some embodiments, the method comprises loading a purified eluate of the protein of interest onto a membrane chromatography support and providing a flow-through containing the further purified eluate from the membrane chromatography support. Further comprising collecting.

In some embodiments, the method does not include an anion exchange chromatography step.

In some embodiments, the method further comprises subjecting the eluate to a virus inactivation step. In some embodiments, virus inactivation is accomplished by low pH virus inactivation to form a virus inactivated preparation. In some embodiments, the pH is reduced to between about 2.5 and about 5. In some embodiments, the method further comprises lowering the pH of the purified eluate of the protein of interest for a period of time sufficient to inactivate the virus.

In some embodiments, the method further comprises filtering the purified eluate of the protein of interest to remove virus.

In some embodiments, the method further comprises formulating the purified eluate of the protein of interest as a composition comprising a pharmaceutically acceptable excipient.

In some embodiments, the method further comprises expressing the protein of interest and one or more contaminating proteins in a bioreactor having a volume of at least about 250 liters. In some embodiments, the bioreactor has a capacity of at least about 500 liters, at least about 2000 liters, or at least about 5000 liters.

In some embodiments, the protein of interest comprises an antibody, or antigen binding fragment thereof, or a fusion protein construct thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises: (a) optionally the antibody or antigen-binding fragment thereof has a VH having the amino acid sequence of SEQ ID NO:5 or 6, and a VL having the amino acid sequence of SEQ ID NO:7. Specifically binding to TNF-like ligand 1A (TL1a), (b) optionally the antibody or antigen-binding fragment thereof has a VH having the amino acid sequence of SEQ ID NO:1 and a VL having the amino acid sequence of SEQ ID NO:2. Specifically binding to calcitonin gene-related peptide (CGRP), or (c) optionally the antibody or antigen-binding fragment thereof has a VH having the amino acid sequence of SEQ ID NO:3, and an amino acid sequence of SEQ ID NO:4. It contains VL and specifically binds to CD38.

In some aspects, provided herein is the use of a wash solution during cation exchange (CEX) chromatography to purify a protein of interest bound to a CEX support, the wash solution comprising: Includes one or more surfactants.

In some embodiments, provided herein is a CEX chromatography wash solution for purifying a protein of interest that is bound to a cation exchange (CEX) support, which comprises a detergent.

In some embodiments of the uses, methods or washes provided herein, the Protein A or CEX chromatography washes have from about 0.1 w/v% to about 0.6 w/v% detergent. including. In some embodiments, the Protein A chromatography wash or CEX chromatography wash comprises from about 0.2 w/v% to about 0.5 w/v% detergent. In some embodiments, the Protein A chromatography wash or CEX chromatography wash comprises about 0.2 w/v% detergent. In some embodiments, the Protein A chromatography wash or CEX chromatography wash comprises about 0.3 w/v% detergent. In some embodiments, the Protein A chromatography wash or CEX chromatography wash comprises about 0.5 w/v% detergent. In some embodiments, the Protein A chromatography wash or CEX chromatography wash comprises about 0.6 w/v% detergent.

In some embodiments of the uses, methods or cleaning provided herein, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is TRITON® X-100. In some embodiments, the wash comprises about 0.2% TRITON® X-100. In some embodiments, the wash comprises about 0.5% TRITON® X-100. In some embodiments, the surfactant is PS80. In some embodiments, the wash comprises about 0.3% PS80. In some embodiments, the wash comprises about 0.6% PS80.

In some aspects of the uses, methods or washes provided herein, the Protein A chromatography wash or CEX chromatography wash further comprises arginine, optionally wherein the Protein A chromatography wash or CEX chromatography wash is , About 25 mM arginine. In some embodiments, the Protein A chromatography wash or CEX chromatography wash further comprises histidine, and optionally the Protein A chromatography wash or CEX chromatography wash comprises about 5 mM histidine. In some embodiments, the Protein A chromatography wash or CEX chromatography wash optionally further comprises sodium chloride and the Protein A chromatography wash or CEX chromatography wash comprises about 5 mM to about 15 mM sodium chloride. In some embodiments, the Protein A chromatography wash or CEX chromatography wash comprises about 10 mM sodium chloride, or about 11 mM sodium chloride. In some embodiments, the Protein A chromatography wash or CEX chromatography wash further comprises sodium phosphate, and optionally the Protein A chromatography wash or CEX chromatography wash comprises about 10 mM sodium phosphate. In some embodiments, the Protein A chromatography wash or CEX chromatography wash further comprises sodium acetate, and optionally the Protein A chromatography wash or CEX chromatography wash comprises about 20 mM to about 50 mM sodium acetate. In some embodiments, the Protein A or CEX chromatography wash comprises about 25 mM sodium acetate, or about 50 mM sodium acetate.

In some embodiments of the uses, methods or washes provided herein, the Protein A chromatography wash or CEX chromatography wash comprises (a) about 10 mM sodium phosphate and about 0.2% TRITON (registered trademark). Trademark) X-100, (b) about 10 mM sodium phosphate and about 0.5% TRITON® X-100, (c) about 10 mM sodium phosphate and about 0.3% PS80, or (D) Containing about 10 mM sodium phosphate and about 0.6% PS80. In some embodiments, the Protein A or CEX chromatography wash comprises (a) about 50 mM sodium acetate, about 25 mM arginine, about 5 mM histidine, about 11 mM sodium chloride and about 0.3% PS80. (B) about 50 mM sodium acetate, about 25 mM arginine, about 5 mM histidine, about 11 mM sodium chloride and about 0.6% PS80, (c) about 50 mM sodium acetate, about 25 mM arginine, about 5 mM Histidine, about 11 mM sodium chloride and about 0.2% TRITON® X-100, (d) about 50 mM sodium acetate, about 25 mM arginine, about 5 mM histidine, about 11 mM sodium chloride and about 0.5% TRITON® X-100, (e) about 25 mM sodium acetate, about 10 mM sodium chloride and about 0.3% PS80, (f) about 25 mM sodium acetate, about 10 mM chloride. Sodium and about 0.6% PS80, (g) about 25 mM sodium acetate, about 10 mM sodium chloride and about 0.2% TRITON® X-100, or (h) about 25 mM sodium acetate, Contains about 10 mM sodium chloride and about 0.5% TRITON® X-100.

In some aspects of the uses, methods or washes provided herein, the Protein A chromatography wash or CEX chromatography wash has a pH of about 5.0 to 7.0. In some embodiments, the Protein A chromatography wash or CEX chromatography wash has a pH of about 5.5 or about 6.3.

In some embodiments, provided herein is a preparation of the protein of interest produced by any of the methods provided herein. In some embodiments, provided herein are viral inactivated preparations of the protein of interest produced by any of the methods provided herein. In some embodiments, the protein of interest is an antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) optionally VH having the amino acid sequence of SEQ ID NO:5 or 6, and the amino acid sequence of SEQ ID NO:7. (B) optionally the antibody or antigen-binding fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO:1, and an amino acid sequence of SEQ ID NO:2, which comprises VL and specifically binds to TNF-like ligand 1A (TL1a) VL, which specifically binds to calcitonin gene-related peptide (CGRP), or (c) optionally the antibody or antigen-binding fragment thereof comprises VH having the amino acid sequence of SEQ ID NO:3, and the amino acid sequence of SEQ ID NO:4. And specifically binds to CD38.

In some embodiments, provided herein is a preparation of a protein of interest produced by any of the methods provided herein, and a pharmaceutically acceptable excipient. It is a composition containing. In some embodiments, provided herein are viral inactivated preparations of a protein of interest produced by any of the methods provided herein, and pharmaceutically acceptable. A composition comprising an excipient.

In some embodiments, provided herein is a composition comprising an aqueous carrier and a recombinantly expressed or hybridoma expressed antibody or antigen binding fragment thereof, the composition being substantially free of viral particles. , An antibody or an antigen-binding fragment thereof (a) specifically binds to TNF-like ligand 1A (TL1a) and has one or more complementarity determining regions (CDRs) of VH having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. And (b) one or more VHs specifically binding to calcitonin gene-related peptide (CGRP) and having one or more CDRs of VL having the amino acid sequence of SEQ ID NO:7 and having the amino acid sequence of SEQ ID NO:1. And one or more CDRs of VL having the amino acid sequence of SEQ ID NO:2, or (c) one or more CDRs of VH having the amino acid sequence of SEQ ID NO:3, which specifically binds to CD38, And one or more CDRs of the VL having the amino acid sequence of SEQ ID NO:4. In some embodiments, the composition is free of viral particles. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) VH having the amino acid sequence of SEQ ID NO:5 or 6, and VL having the amino acid sequence of SEQ ID NO:7, (b) of SEQ ID NO:1 VH having an amino acid sequence and VL having the amino acid sequence of SEQ ID NO:2, or (c) VH having the amino acid sequence of SEQ ID NO:3 and VL having the amino acid sequence of SEQ ID NO:4.

In some embodiments, provided herein are: a) loading onto Protein A chromatography support a mixture comprising antibody and one or more contaminants, aggregates and/or viruses, b) washing the support with a Protein A chromatography wash containing one or more detergents to remove one or more contaminating proteins, aggregates and/or viruses from the Protein A chromatography support. C) eluting the antibody from a protein A chromatography support, d) loading the eluate from step c) onto a cation exchange (CEX) chromatography support, e) one or more surfactants. Washing the CEX chromatographic support with a CEX chromatographic wash solution containing a. to remove one or more contaminating proteins, aggregates and/or viruses from the support, and f) eluting the antibody from the CEX chromatographic support. A composition comprising an aqueous carrier prepared by the method and a recombinantly expressed or hybridoma-expressed antibody, the method comprising forming a purified eluate of the antibody. In some embodiments, the antibody or antigen-binding fragment thereof (a) specifically binds to TNF-like ligand 1A (TL1a) and is complementary to one or more of VH having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. (B) specifically binding to a calcitonin gene-related peptide (CGRP), comprising a sex determining region (CDR) and one or more CDRs of VL having the amino acid sequence of SEQ ID NO:7, Of VH having one or more CDRs of VH and one or more CDRs of VL having the amino acid sequence of SEQ ID NO:2, or (c) specifically binding to CD38 and having the amino acid sequence of SEQ ID NO:3. One or more CDRs and one or more CDRs of a VL having the amino acid sequence of SEQ ID NO:4. In some embodiments, the composition is substantially free of viral particles. In some embodiments, the composition has not undergone anion exchange chromatography prior to CEX chromatography. In some embodiments, the composition has not undergone any ion exchange chromatography prior to CEX chromatography.

1 is a schematic of an exemplary protein purification process using the methods provided herein. As demonstrated in the working examples, a "detergent wash" is capable of substantially removing viral particles and also yields as compared to using a detergent-free wash. Can be increased. "Wash 3", the intermediate wash step, does not include a wash step to remove the detergent by removing the detergent and instead requires discarding the initial elution fraction containing the detergent. It is possible to increase the yield compared to the process. These methods outlined in this schematic can be used without a Protein A purification step or in combination with a Protein A purification step (eg, downstream).

It was observed according to the present disclosure that the inclusion of a detergent in the CEX wash liquor substantially enhanced virus clearance from the protein preparation during CEX chromatographic purification. This is advantageous as it allows omitting the AEX chromatography step in the protein purification method. Surprisingly, it was found that the surfactant had no deleterious effects on the protein of interest. Furthermore, it was found that CEX washes increased the removal of host cell-derived proteins from protein preparations. Also, after the detergent-containing wash, but before elution of the protein from the CEX chromatography support, the use of an intermediate wash removes any residual detergent from the CEX chromatography support, in addition to the elution. It was found that the yield of the protein of interest in the solution was surprisingly improved and the HCP exclusion was improved.

It was observed that the levels of HCP removal and virus clearance were considerable, so that subsequent purification steps by anion exchange (AEX) chromatography could be omitted from the protein purification scheme. Therefore, in one embodiment, the protein purification method described herein does not include an AEX step. Such protein purification methods can include an affinity chromatography step followed by a CEX chromatography step as described herein.

The present disclosure has been purified by affinity and CEX chromatography wash compositions, protein purification schemes utilizing compositions such as these, and having a high degree of purity, eg, using compositions or purification schemes such as these. Such protein preparations are characterized. The compositions and purification schemes are particularly well suited for hybridoma-expressing or recombinantly expressing monoclonal antibodies, but can also be used for preparation of any recombinantly expressed protein purified by CEX chromatography.

Accordingly, provided herein are affinity and cation exchange chromatography wash compositions, polypeptide purification schemes utilizing such compositions, and compositions having high purity, eg, compositions such as these. Or a polypeptide preparation, as purified by use of a purification scheme.

I. Definitions Throughout this specification and the claims, various terms relating to aspects of the present invention are used. Unless otherwise indicated, such terms shall be given their ordinary meaning in the art. Other terms specifically defined shall be construed in a manner consistent with the definitions set forth herein.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

The term "solution containing water" is used interchangeably with the term "aqueous solution".

The terms "host cell-derived protein", "HCP", "host cell-derived protein contaminant" and "host cell-derived protein impurity" are used interchangeably herein.

The terms “polypeptide”, “peptide”, “polypeptide of interest”, and “protein” are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. These terms have been modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling moiety. Also included are amino acid polymers. Also included in this definition are, for example, polypeptides that include one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), and other modifications known in the art. It is understood that since the polypeptides of the present disclosure are antibody-based, in certain embodiments, the polypeptides can exist as single chains or binding chains. Preferably, the protein of interest is an antibody, or antigen-binding fragment thereof, or antibody construct.

The term "anti-TNF-like ligand 1A (TL1a)" refers to any protein capable of binding TL1a. Anti-TL1a proteins include, for example, anti-TL1a antibodies, or antigen binding fragments thereof. The antibody can be any of the antibodies described in US Publication No. 2014/0255302, which is incorporated herein by reference. The antibody can be any of the antibodies described in US Provisional Application No. 62/220,442.

The term "anti-calcitonin gene related peptide (CGRP)" refers to any protein capable of binding CGRP. Anti-GCRP proteins include, for example, anti-CGRP antibodies, or antigen binding fragments thereof. The antibody can be any of the antibodies described in US Publication No. 2009/0220489 or PCT Publication No. WO2007/054809.

The term "anti-CD38" refers to any protein capable of binding CD38. Anti-CD38 proteins include, for example, anti-CD38 antibodies, or antigen binding fragments thereof. The antibody can be any of the antibodies described in US Publication No. 2016/0068612 or US Publication No. 2015/0313965, each of which is incorporated herein by reference and these It includes an antibody further fused to an attenuated interferon molecule as described in the publication.

As used herein, the terms “antibody” and “antibody” are technical terms and can be used interchangeably herein and are specific for an antigen. Refers to a molecule containing an antigen binding site that binds to.

The term “antibody” recognizes a target, eg, a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof via at least one antigen recognition site within the variable region of the immunoglobulin molecule. And an immunoglobulin molecule that binds to it. As used herein, the term "antibody" refers to an intact polyclonal antibody, an intact monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein comprising an antibody, and the desired biological activity of the antibody. Any other modified immunoglobulin molecule is meant to be shown. Antibodies are based on the identity of heavy chain constant domains called alpha, delta, epsilon, gamma, and mu, respectively, in five major immunoglobulin classes, namely IgA, IgD, IgE, IgG, and IgM, or It can be any of those subclasses (isotypes) (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Different classes of immunoglobulins have different well-known subunit and three-dimensional structures. The antibody may be as it is or may be bound to other molecules such as toxins and radioisotopes. The term “antibody” as used herein includes bispecific and multispecific antibodies.

The term "antibody fragment" refers to a portion of an intact antibody. "Antigen-binding fragment" refers to the portion of an intact antibody that binds an antigen. The antigen-binding fragment may comprise the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single chain antibodies. An "antigen-binding fragment" can be a bispecific or multispecific antigen binding fragment.

The term "monoclonal" antibody or antigen-binding fragment thereof refers to a single antigenic determinant, ie a homogeneous population of antibodies or antigen-binding fragments involved in the highly specific recognition and binding of epitopes. This is in contrast to polyclonal antibodies which usually contain different antibodies against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof refers to antibody fragments (eg, Fab, Fab', F(ab')2, Fv), single chain (scFv) variants, as well as intact and full-length monoclonal antibodies. , A fusion protein containing an antibody portion, and any other immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal" antibody or antigen binding fragment thereof refers to antibodies and antigen binding fragments thereof produced by any method, including but not limited to hybridoma, phage selection, recombinant expression, and transgenic animals.

The term "humanized" antibody or antigen-binding fragment thereof refers to a particular immunoglobulin chain that contains minimal non-human (eg, murine) sequences, chimeric immunoglobulins, or fragments thereof, non-human (eg, murine). Refers to an antibody or antigen-binding fragment. Generally, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin in which the residues from its complementarity determining regions (CDRs) have a desired specificity, affinity, and function (eg, , Mouse, rat, rabbit, hamster) have been replaced with residues derived from CDRs ("CDR graft") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-. 327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, the Fv framework region (FR) of human immunoglobulin is replaced with the corresponding residue in an antibody or fragment from a non-human species that has the desired specificity, affinity, and function. The humanized antibody or antigen-binding fragment thereof is further modified by substitution of additional residues within the Fv framework regions and/or replaced non-human residues to provide specificity, affinity of the antibody or antigen-binding fragment thereof, And/or functionality can be improved and optimized. Generally, a humanized antibody or antigen-binding fragment thereof comprises substantially all of at least one, usually two or three variable domains, comprising all or substantially all of the CDR regions corresponding to non-human immunoglobulin. However, all or substantially all of the FR regions are of the human immunoglobulin consensus region. The humanized antibody or antigen binding fragment thereof may also include at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in US Pat. No. 5,225,539, Roguska et al. , Proc. Natl. Acad. Sci. , USA, 91(3):969-973 (1994), and Roguska et al. , Protein Eng. 9(10):895-904 (1996). In some embodiments, "humanized antibody" is a resurfaced antibody.

The “variable region” of an antibody refers to the variable regions of an antibody light chain or the variable regions of an antibody heavy chain, alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions joined by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs within each chain are held together in close proximity by the FRs and, along with the CDRs from the other chains, contribute to the formation of the antigen-binding site of the antibody. At least two techniques for determining CDRs, ie, (1) methods based on cross-species sequence variation (ie Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Health). Md.)), and (2) a method based on a crystallographic study of an antigen-antibody complex (Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)). Furthermore, the art may use a combination of these two methods to determine the CDR.

The term "human antibody" means an antibody that has a amino acid sequence corresponding to a human-produced antibody, or a human-produced antibody that is produced using any technique known in the art. This definition of human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy chain and/or light chain polypeptide, eg, mouse light chain and human heavy chain polypeptides. Including antibodies and the like.

The term "hybridoma expression" refers to the protein of interest expressed in a hybrid cell line produced by the fusion of an immortalized cell line of immunological origin and an antibody producing cell. The term "hybridoma" refers to the progeny of a heterohybrid myeloma fusion, which is the result of the fusion of human cells and mouse myeloma cell lines, which are subsequently fused with plasma cells, commonly known as trioma cell lines. Include. In addition, the term "hybridoma" is meant to include any immortalized hybrid cell line that produces antibodies, such as quadroma (see, eg, Milstein et al., 1983, Nature, 537:3053). .. The hybrid cell line can be of any species, including human and mouse.

The term "recombinant expression" refers to a subject expressed in a "recombinant host cell" that has been genetically modified or can be genetically modified by the introduction of an exogenous polynucleotide such as a recombinant plasmid or vector. Refers to the protein. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such cell. Although certain modifications may occur in subsequent generations, either due to mutations or environmental influences, such progeny may not be identical to the parent cell in nature, but herein. Still included within the scope of the term "recombinant host cell" as used in.

The term "amino acid" refers to naturally occurring and/or unnatural amino acid residues. The term "naturally occurring amino acid" refers to Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. Point to. The term "basic amino acid" refers to arginine, lysine, glycine and histidine. Amino acids also include the D-forms of natural and unnatural amino acids. "D-" indicates an amino acid having the "D" (dextrorotatory) configuration as opposed to the configuration in the naturally occurring ("L-") amino acid. Natural and unnatural amino acids may be obtained commercially (Sigma Chemical Co., Advanced Chemtech) or may be synthesized using methods known in the art.

The term "virus clearance" is used interchangeably with the terms "virus removal" and "virus removal". The term "virus inactivation" refers to rendering the viruses contained in a mixture non-functional. Viruses can originate from sources of antibody production, downstream processing steps or manufacturing conditions. Methods of rendering the virus non-functional or removing the virus include heat activation, pH inactivation, chemical inactivating agents and the like. The term "pH virus inactivation" includes exposing the virus to a pH sufficient to render the virus non-functional, eg, a pH between about 2.5 and 5.0.

The terms "log ₁₀ reduction factor (LRF)", "log ₁₀ reduction value (LRV)", and "log clearance" are interchangeable and viral titers in starting material and in related product fractions. Refers to the calculated ratio of. The reduction factor is a suitable parameter that describes the potential or ability of a process step to remove or inactivate a virus. The LRV of either process step uses any known model virus that resembles a virus that can contaminate the product, eg, murine leukemia virus (MuLV) and mouse microvirus (MVM). Can be measured. Also, LRV can be measured by retrovirus-like particles (RVLP). LRV can be calculated by quantitative PCR (qPCR) or using an infection assay (eg, measuring TCID ₅₀ ).

The present invention provides a purified eluate and composition comprising a protein of interest that is "substantially free" of viral particles, as measured by virus exclusion studies using any of the methods of the invention. provide. As used herein, the term "substantially free of viral particles" refers to purified eluates and compositions containing the protein of interest, wherein the protein of interest has been separated from the viral particles. .. The term "substantially free" refers to a solution or composition containing a protein of interest containing less than about 0.0005% to less than about 0.001% viral particles. Preferably, the composition is "substantially free" when it comprises less than about 0.0005% viral particles.

The present invention provides purified eluates and compositions containing proteins of interest that are “free” of viral particles, as measured by virus exclusion studies using any of the methods of the invention. As used herein, the term "free of viral particles" refers to compositions having less than about 0.0001%. The composition is virus particle free when the virus particles cannot be detected by virus exclusion studies under the most sensitive conditions.

The terms "column", "support", "ligand", and "resin" are used interchangeably herein.

The terms "composition" and "purified composition" are interchangeable and refer to a composition comprising a carrier and a polypeptide of interest. This carrier is preferably water-soluble and can be a pharmaceutically acceptable carrier. The carrier can include buffers and can include one or more pharmaceutically acceptable excipients. This composition can be referred to as a pharmaceutical composition.

The term "affinity chromatography" or "affinity purification" refers to a separation method based on specific binding interactions between a ligand immobilized on or bound to a solid support and its binding partner. As the complex mixture passes through the column, those molecules that have a specific binding affinity for the ligand become bound. After washing off the other sample composition, the bound molecules are removed from the support, resulting in its purification from the original mixture. Each specific affinity system requires its own set of conditions, known to those skilled in the art.

The term "affinity ligand" refers to metals (eg, Cd+2, Co+2, Cu+2, Ga+3, Fe+3, Ni+2, and Zn+2), pigments (eg, Cibacron Blue and its variants), glutathione, subtilisin, protein A, protein. G, protein A/G, protein L, boronate, avidin, streptavidin, biotin, anti-c-Myc, anti-HA, nucleotides, coenzymes, antibodies, heparin, antigens (in particular, antibodies having known specificity) ), and other known affinity ligands.

II. Compositions Comprising Proteins of Interest and Their Preparations for Purification As demonstrated herein, methods of purifying proteins include the use of detergent-containing washes (eg, CEX chromatography column washes). Can be improved through. Using these methods, it is possible to capture and purify recombinantly expressed proteins directly, for example from harvested cell culture fluid (HCCF). It is also possible to use these methods to further purify recombinantly expressed proteins, eg proteins which have already been partially purified (eg via protein A purification) from HCCF. is there.

In some embodiments, the protein of interest preferably comprises an antibody or antigen binding fragment thereof. In some embodiments, the protein of interest is an antibody that specifically binds to TNF-like ligand 1A (TL1a). An antibody that specifically binds to TL1a is a heavy chain variable region (VH) that comprises the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6 (or a sequence having 90%, 95%, or 99% identity thereto). , And a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 7 (or a sequence having 90%, 95%, 95%, or 99% identity thereto). In some embodiments, the protein of interest is an antibody that specifically binds to calcitonin gene-related peptide (CGRP). An antibody that specifically binds to CGRP comprises a VH having the amino acid sequence of SEQ ID NO: 1 (or a sequence having 90%, 95%, or 99% identity thereto), and the amino acid sequence of SEQ ID NO: 2 (or VL with 90%, 95%, or 99% sequence identity thereto. In some embodiments, the protein of interest is an antibody that specifically binds CD38. Antibodies that specifically bind to CD38 include VH having the amino acid sequence of SEQ ID NO:3 (or a sequence having 90%, 95%, or 99% identity thereto), and the amino acid sequence of SEQ ID NO:4 (or VL with 90%, 95%, or 99% sequence identity thereto. Antibodies that specifically bind to CD38 include interferon alpha molecules and can further include fusions to interferon molecules, including attenuated interferon alpha molecules.

Expression of the protein of interest can be carried out in any suitable host cell that can be transformed with the gene encoding the protein. Host cells can be eukaryotic or prokaryotic, including but not limited to bacterial cells, yeast cells, insect cells, and mammalian cells. Mammalian cells are preferred. Non-limiting examples of suitable mammalian cells include antibody expressing hybridoma cells as well as expression hosts such as Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, and mouse hybridoma NS0 cells. The expressed polypeptide can be secreted from the cell into the cell culture medium or can be intracellular. Cell culture can be performed in a bioreactor (eg, fermentation). A typical bioreactor cell culture is started with a basal medium, after which the nutrients are regularly infused until the completion of the culture. This infusion, which is typically that of the feed medium, maintains the cell culture during the protein expression phase. Roughly, feed medium injection is performed via bolus injection, and concentrated feed medium is usually added once a day into cell culture medium at set time points. As an alternative to a bolus feed, the bioreactor cell culture fluid can be infused using an extended or continuous feed. Commercially available feed media are suitable for bioreactor nutrient infusion.

The bioreactor can have a capacity of at least about 250 liters. In some embodiments, the bioreactor has a capacity of at least about 500 liters. In some embodiments, the bioreactor has a capacity of at least about 2000 liters. In some embodiments, the bioreactor has a capacity of at least about 5000 liters, or 10,000 liters, or 15,000 liters.

After expression, the medium containing the polypeptide (eg, cell culture medium) can be clarified, eg, to remove host cells and particulate debris. Clarification can include filtration, centrifugation, or a combination thereof. For example, depth filtration through diatomaceous earth and cellulosic fibers can be used. Membrane filtration can be used to remove any microbial contaminants using any commercially available membrane filter, for example through a 0.2 μm filter.

When used after expression and clarification, the polypeptide of interest can be purified via affinity chromatography to remove contaminating host cell-derived protein (HCP). Affinity chromatography can include any affinity ligand suitable for purification of the polypeptide of interest. Non-limiting examples of affinity chromatography ligands include metals (eg, Cd ⁺² , Co ⁺² , Cu ⁺² , Ga ⁺³ , Fe ⁺³ , Ni ⁺² , and Zn ⁺² ), dyes (eg, Cibacron Blue and Its variant), glutathione, subtilisin, protein A, protein G, protein A/G, protein L, boronate, avidin, streptavidin, biotin, anti-c-Myc, anti-HA, nucleotides, coenzymes, antibodies, heparin, Includes antigens (especially for antibodies of known specificity), and other known affinity ligands. The affinity ligand is typically immobilized on a solid support, which has a number of known and common supports. In one embodiment of the invention, CEX chromatography with the wash solutions described herein can replace the steps of affinity chromatography. In this embodiment, when used after expression and clarification, an aqueous solution containing the polypeptide of interest can be loaded onto the CEX support without prior affinity chromatography.

III. After CEX purification affinity chromatography (when used), the protein of interest is bound to this support by loading the protein preparation onto the CEX support. This support has a high protein binding capacity. The support is preferably equilibrated before loading with the polypeptide preparation. Equilibration is preferably done with a buffer.

In some embodiments, the CEX support is Sepharose matrix resin. In some embodiments, the CEX support is a synthetic polymer matrix resin. Exemplary CEX supports include GE Capto S, Millipore Fractogel EMD SO3-, Fractogel EMD TMAE, Fractogel EMD, Eshnumo S, Eshmuno porpoi HCM, Tosoh Toyopoorpoyl, and Tosoh Toyopoorpoyl.

In some embodiments, the CEX support is a sepharose-based strong cation exchange of sulfopropyl. The CEX support can be, for example, SPFF resin.

In some embodiments, the CEX support comprises a poly(styrenedivinylbenzene) polymer matrix crosslinked with sulfopropyl ligand. The CEX support can be, for example, Poros XS resin.

In some embodiments, the CEX support is a polymeric ion exchange chromatography resin (Poros™ XS, Life Technologies Corp., Carlsbad, CA).

In some embodiments, the CEX support is 6% agarose crosslinked by a strong anion exchange group of quaternary ammonium (Q) (Q Sepharose® Fast Flow, GE-Healthcare, Pittsburgh, PA). The beads.

Loading of a polypeptide preparation onto a CEX chromatography support is carried out at a temperature, in volume, and at a temperature suitable to allow adsorption of the polypeptide of interest onto the support. Unwanted HCPs and viruses that do not bind to the support flow through the support during chromatography. The wash fluids described and exemplified herein are used to wash CEX chromatography supports for removal of viruses, aggregates and contaminants including HCPs. After washing with the intermediate wash buffer, the protein of interest is eluted from the CEX support. The elution buffer is generally tailored to the type of CEX support and polypeptide of interest, and, as a result, can be different. Elution can be performed at a temperature, in volume, and at a time suitable to allow maximum elution yield of the protein of interest. Elution of this protein produces a CEX chromatography eluate containing this protein. Elution of the antibody or antibody construct produces a CEX eluate containing the antibody or antibody construct.

While reducing HCP and aggregate content, the CEX chromatography washes of the present invention can be used to reduce virus levels or inactivate viruses in compositions containing the protein of interest. .. Typical prior art purification processes use anion exchange followed by a further acidification treatment to reduce virus levels or inactivate the virus during protein (eg, antibody) purification methods. Surprisingly, the inventors have found that the use of the CEX chromatography wash of the present invention reduces the level of virus sufficient to omit the anion exchange chromatography step, or the virus inactivation step. ..

IV. Prior to loading the viral exclusion protein onto the CEX chromatography support using detergent washes and other methods, the protein solution (eg, containing the polypeptide, or containing the antibody or antibody construct) is in the eluate. Can be further treated by a treatment that inactivates any residual virus present in. Virus inactivation can include acidifying the eluate for a period of time at a temperature sufficient to inactivate any virus present in the eluate. This acidification may include, for example, adding acetic acid, citric acid, hydrochloric acid, formic acid, phosphoric acid, caprylic acid, other suitable acids, or combinations thereof to the eluent until the desired pH is achieved. it can. After low pH virus inactivation, the eluate can be neutralized to pH 3.0 to 7.5 (depending on process needs). In some embodiments, the eluent is a pH of about 3.0 to about 3.5, a pH of about 3.0 to about 4.0, a pH of about 3.5 to about 4.5, about 4. It is neutralized to a pH of 0 to about 5.5, a pH of about 4.5 to 5.5, a pH of about 6.0 to about 7.0, or a pH of about 6.0 to about 7.5. In some embodiments, the eluent is a pH of about 3.0, a pH of about 3.5, a pH of about 4.0, a pH of about 4.5, a pH of about 5.0, about 5. Neutralized to a pH of 5, about 6.0, about 6.5, about 7.0 or about 7.5. During the neutralization step, turbidity may be found in the product pool due to precipitation of impurities (or products). Deep filtration can be used to filter the pH adjusted preparation to remove turbidity and impurities.

A unit operation to inactivate viruses present in a fluid containing the protein of interest can be carried out in a holding tank, the holding tank being between about 2.5 and 5.0, Between about 3.5 and about 4.5, between about 3.5 and about 4.25, between about 3.5 and about 4.0, between about 3.5 and about 3.8 Or at a pH of about 3.75 for at least 25 minutes, about 30 minutes to 1.5 hours, about 30 minutes to 1.25 hours, about 0.75 hours to 1.25 hours, or about. The fluid containing the recombinant therapeutic protein can be incubated for 1 hour.

In alternative embodiments, the virus inactivation step can be performed using other methods known in the art. For example, the virus inactivation step is, in various embodiments, acid, detergent, chemical, nucleic acid crosslinker, UV light, gamma radiation, heating, or any known in the art that is useful for this purpose. Processing by any other process may be included.

The virus can be removed by filtration. For example, viral filtration can be performed before and/or after the step of flushing the recombinant protein through the depth filter. Viruses are removed from solutions containing recombinant proteins by either normal flow filters (NFF) or tangential flow filtration (TFF) filters, as described in US Pat. No. 6,365,395. It is possible. Conditions to retain a virus on the surface of this membrane, generally having a diameter of 20 to 100 nanometers (nm), while allowing the passage of recombinant proteins through the membrane in either the TFF or NFF modes. Filter below.

The purpose of virus clearance studies is to evaluate the process step(s) that can be considered effective in inactivating/eliminating the virus, and the virus obtained by the process step(s). It is to quantitatively estimate the overall level of reduction. Removal or inactivation of the virus during subsequent steps after obtaining a level of virus reduction by adding a significant amount of virus (“addition”) to the mixture containing the protein of interest obtained after various process steps Can be demonstrated. Reduction of viral infection can be achieved by removal of viral particles or inactivation of viral infection. Virus exclusion studies are performed to demonstrate the elimination of viruses known to be present in the mixture. The reduction factor is usually expressed on a logarithmic scale (log ₁₀ ). The model viruses for exclusion assessment studies are selected to resemble viruses that may contaminate the mixture containing the protein of interest. Model viruses, such as xenotropic murine leukemia virus (X-MulV) and mouse microvirus (MVM), are often used for virus clearance validation of proteins of interest from cell lines.

In some embodiments, the CEX or ProA washes increase virus clearance or inactivate viruses from the mixture containing the protein of interest. In some embodiments, the wash solution increases viral clearance during the Protein A chromatography step. In some embodiments, the wash solution increases viral clearance during the CEX chromatography step. Viral clearance can be measured at the log ₁₀ reduction value (LRV). In some embodiments, the wash solution increases viral clearance with an LRV of between about 1.0 and about 10.0 log ₁₀ . In some embodiments, the cleaning liquid, LRV is between about 1.0 to about 5.0 log _10, LRV is between about 1.0 to about 3.0 log _10, LRV of about 2.0 To about 4.0 log ₁₀ , LRV between about 3.0 and about 5.0 log ₁₀ , or LRV between about 5.0 log ₁₀ and 6.0 log ₁₀ increase viral clearance Let In some embodiments, LRV is about 1.0 log _10, LRV is about 2.0 log _10, LRV is about 3.0 log _10, LRV is about 4.0Log ₁₀ , LRV is about 5.0 log ₁₀ , LRV is about 6.0 log ₁₀ , LRV is about 7.0 log ₁₀ , LRV is about 8.0 log ₁₀ , LRV is about 9 0.0 log ₁₀ or the LRV is about 10.0 log ₁₀ . LRV can be achieved by applying 1 column volume (CV) wash, 2 CV wash, 3 CV wash, 4 CV wash, or 5 CV wash.

V. The protein of interest after further treatment of the protein purified using a detergent-containing wash, and the composition produced therefrom after virus inactivation, or elution from CEX chromatography without virus inactivation. Can be further processed into a form suitable for therapeutic administration, eg, to a non-human animal, or to a human. Such further processing can include any combination of ultrafiltration, nanofiltration, concentration, and diafiltration of a purified preparation of the protein of interest.

Ultrafiltration is a process for concentrating a preparation of proteins of interest. Filter proteins from other molecules in solution based on membrane pore size or molecular weight cutoff. Diafiltration is used to exchange the protein of interest for the desired buffer (eg, elution buffer into stable formulation buffer). Ultrafiltration and diafiltration generally use tangential flow filtration.

After the purification scheme with the use of the CEX chromatography wash buffer of the invention, the protein of interest is preferably present in the composition. The composition preferably comprises a carrier and the protein of interest. This carrier is preferably water-soluble and can be a pharmaceutically acceptable carrier. The carrier can include buffers and can include one or more pharmaceutically acceptable excipients. This composition is sometimes referred to as a pharmaceutical composition. The terms "composition" and "purified composition" are used interchangeably herein.

Provided in some embodiments is a composition comprising a purified protein of interest, wherein the protein is purified according to the methods described herein or illustrated. Compositions such as these contain the protein of interest and a minimal amount of retrovirus-like particles (RVLPs), the latter being co-expressed with the protein of interest, but using the wash solution described herein. Almost separated from the protein of interest via CEX chromatography.

In some embodiments, the polypeptide of interest in the composition is an antibody, or antibody construct. The antibody or antibody construct can be expressed recombinantly by a transformed host cell (eg, a host cell that contains a gene encoding the antibody or antibody construct) or can be expressed via a hybridoma cell. .. The antibody or antibody construct is capable of specifically binding an epitope on human TNF-like ligand 1A (TL1a). The antibody or antibody construct can specifically bind to an epitope on human calcitonin gene related peptide (CGRP). The antibody or antibody construct is capable of specifically binding an epitope on human CD38.

In some embodiments, the composition comprises a purified antibody or antibody construct that specifically binds CGRP and comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region (VH) can have the amino acid sequence of SEQ ID NO:1. The light chain variable region (VL) can have the amino acid sequence of SEQ ID NO:2. The composition can include an antibody or antibody construct comprising VH and VL having the amino acid sequence of SEQ ID NO: 1, which antibody or antibody construct specifically binds CGRP. The composition can include an antibody or antibody construct comprising VL having the amino acid sequence of SEQ ID NO:2, and VH, which antibody or antibody construct specifically binds CGRP. The composition can include an antibody or antibody construct that comprises a VH having the amino acid sequence of SEQ ID NO:1 and a VL having the amino acid sequence of SEQ ID NO:2, which antibody or antibody construct specifically binds to CGRP. .. The antibody can be any of the antibodies described in US Publication No. 2009/0220489 or PCT Publication No. WO2007/054809.

In some embodiments, the composition comprises a purified antibody or antibody construct that specifically binds TL1a and comprises a heavy chain variable region and a light chain variable region. The antibody can be any of the antibodies described in US Publication No. 2014/0255302, which is incorporated herein by reference. The antibody can be any of the antibodies described in US Provisional Application No. 62/220,442.

In some embodiments, the composition comprises a purified antibody or antibody construct that specifically binds CD38 and comprises a heavy chain variable region and a light chain variable region. The anti-CD38 antibody can be further fused to a second polypeptide molecule, eg, fused to a polypeptide toxin, or fused to an interferon polypeptide such as interferon alpha. The heavy chain variable region (VH) can have the amino acid sequence of SEQ ID NO:3. The light chain variable region (VL) can have the amino acid sequence of SEQ ID NO:4. The composition can include an antibody or antibody construct comprising VH and VL having the amino acid sequence of SEQ ID NO:3, which antibody or antibody construct specifically binds to CD38. The composition can include an antibody or antibody construct containing VL and VH having the amino acid sequence of SEQ ID NO:4, which antibody or antibody construct specifically binds to CD38. The composition can include an antibody or antibody construct comprising a VH having the amino acid sequence of SEQ ID NO:3 and a VL having the amino acid sequence of SEQ ID NO:4, which antibody or antibody construct specifically binds to CD38. .. The antibody can be any of the antibodies described in US Publication No. 2016/0068612 or US Publication No. 2015/0313965, each of which is incorporated herein by reference and these It includes an antibody further fused to an attenuated interferon molecule as described in the publication.

In some embodiments, the composition comprises a purified antibody or antibody construct that specifically binds TL1a and comprises a heavy chain variable region and a light chain variable region. The VH can have the amino acid sequence of SEQ ID NO:5. The VH can have the amino acid sequence of SEQ ID NO:6. The VL can have the amino acid sequence of SEQ ID NO:7. Thus, a VH can include SEQ ID NO:5, a VL can include SEQ ID NO:7, or a VH can include SEQ ID NO:6, and a VL can include SEQ ID NO:7. .. The composition can include an antibody or antibody construct comprising VH and VL having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, which antibody or antibody construct specifically binds to TL1a. The composition can include an antibody or antibody construct containing VL having the amino acid sequence of SEQ ID NO:7 and VH, the antibody or antibody construct specifically binding to TL1a. The composition can include a VH having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6, and a VL-containing antibody or antibody construct having the amino acid sequence of SEQ ID NO:7, the antibody or antibody construct specific for TL1a. Join together. The antibody can be any of the antibodies described in US Application No. 15/267,213 or US Publication No. 2014/0255302, each of which is hereby incorporated by reference. ..

In some preferred embodiments, these antibodies (eg, anti-TL1a, anti-CGRP, and anti-CD38) include human IgG constant regions. The human IgG constant region can be a human IgG or human IgG4 constant region. These antibodies (eg, anti-TL1a, anti-CGRP, and anti-CD38) can be humanized antibodies or fully human antibodies.

VI. Wash Solution A wash solution containing a detergent, as provided herein, can be used in a method for purifying proteins. A detergent-containing wash solution is surprisingly effective at removing viruses. Washes such as these can be used, for example, in cation exchange chromatography, which may or may not be advanced by the Protein A chromatography step. The Protein A chromatography step can also use a detergent-containing wash, or can be performed with a detergent-free wash.

The surfactant in the wash liquor can be, for example, a nonionic surfactant. Examples of the surfactant in the washing liquid include CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid), CAHS (coconut oil fatty acid amide propylhydroxysultaine) or SB-12N-. Zwitterionic surfactants such as dodecyl-N,N-dimethylammonio-3-propanesulfonic acid; quaternary ammonium salts such as CTAB (cetrimonium bromide) or DODAB (dioctadecyldimethylammonium bromide); nonoxynol (eg, , Ethoxylates; alkyl polyglucosides such as decyl glucosides, amines or phosphine oxides; and/or anionic surfactants such as SDS (sodium dodecyl sulphate).

Examples of the surfactant in the cleaning liquid include Triton 100, nonylphenoxypolyethoxylethanol (NP40), sulfobetaine-12 (SB-12), sulfobetaine-14 (SB-14), lauryldimethylamine N-oxide (LDAO). , Polysorbate 20 (PS20), Polysorbate 80 (PS80), or a combination thereof.

The detergent in the wash liquor can be, for example, Triton 100, NP40, LDAO, SB-12, and/or SB-14.

In one particular example, the wash liquor has a predetermined concentration of surfactant. The concentration of surfactant in the wash liquor can be, for example, from about 0.01 w/v% to about 1 w/v%. The concentration of surfactant in the wash liquor can be, for example, about 0.05 w/v% to about 1 w/v%. The concentration of surfactant in the wash liquor can be, for example, from about 0.1 w/v% to about 1 w/v%. The concentration of surfactant in the wash liquor can be, for example, about 0.2 w/v% to about 1 w/v%. The concentration of surfactant in the wash liquor can be, for example, about 0.5 w/v% to about 1 w/v%.

The concentration of surfactant in the wash liquor can be, for example, about 0.01 w/v% to about 0.5 w/v%. The concentration of surfactant in the wash liquor can be, for example, from about 0.05 w/v% to about 0.5 w/v%. The concentration of surfactant in the wash liquor can be, for example, from about 0.1 w/v% to about 0.5 w/v%. The concentration of surfactant in the wash liquor can be, for example, about 0.2 w/v% to about 0.5 w/v%.

The concentration of surfactant in the wash liquor can be, for example, about 0.01 w/v %. The concentration of surfactant in the wash liquor can be, for example, about 0.05 w/v %. The concentration of surfactant in the wash liquor can be, for example, about 0.1 w/v %. The concentration of surfactant in the wash liquor can be, for example, about 0.2 w/v %. The concentration of surfactant in the wash liquor can be, for example, about 0.5 w/v %. The concentration of surfactant in the wash liquor can be, for example, about 1 w/v %.

In addition to the detergent, the wash solution can include, for example, sodium acetate (eg, at a concentration of 25 mM) and/or sodium chloride (eg, at a concentration of 10 mM). Such a cleaning liquid can have a pH of, for example, about 5.5.

In addition to the detergent, the wash solution can include, for example, sodium phosphate (eg, at a concentration of 10 mM). Such a wash solution can have a pH of, for example, about 6.3.

In one embodiment, the wash solution contains 10 mM sodium phosphate and 0.2% TRITON® X-100.

In one embodiment, the wash solution comprises 10 mM sodium phosphate and 0.5% TRITON® X-100.

In one embodiment, the wash solution comprises 10 mM sodium phosphate and 0.3% PS80.

In one embodiment, the wash solution contains 10 mM sodium phosphate and 0.6% PS80.

In one embodiment, the wash solution contains 10 mM sodium phosphate and 0.2% TRITON® X-100.

In one embodiment, the wash solution comprises 10 mM sodium phosphate and 0.5% TRITON® X-100.

In one embodiment, the wash solution comprises 10 mM sodium phosphate and 0.3% PS80.

In one embodiment, the wash solution contains 10 mM sodium phosphate and 0.6% PS80.

In one embodiment, the wash solution comprises 50 mM sodium acetate, 25 mM arginine, 5 mM histidine, 11 mM sodium chloride and 0.3% PS80.

In one embodiment, the wash solution comprises 50 mM sodium acetate, 25 mM arginine, 5 mM histidine, 11 mM sodium chloride and 0.6% PS80.

In one embodiment, the wash solution contains 50 mM sodium acetate, 25 mM arginine, 5 mM histidine, 11 mM sodium chloride and 0.2% TRITON® X-100.

In one embodiment, the wash solution contains 50 mM sodium acetate, 25 mM arginine, 5 mM histidine, 11 mM sodium chloride and 0.5% TRITON® X-100.

In one embodiment, the wash solution contains 25 mM sodium acetate, 10 mM sodium chloride and 0.3% PS80.

In one embodiment, the wash solution comprises 25 mM sodium acetate, 10 mM sodium chloride and 0.6% PS80.

In one embodiment, the wash solution comprises 25 mM sodium acetate, 10 mM sodium chloride and 0.2% TRITON® X-100.

In one embodiment, the wash solution contains 25 mM sodium acetate, 10 mM sodium chloride and 0.5% TRITON® X-100.

In one embodiment, the disclosure features a CEX chromatography solution that includes one or more surfactants. In some embodiments, the disclosure features a CEX chromatography wash that includes a basic amino acid, a salt, a nonionic detergent or buffer (eg, an organophosphate), and a detergent. .. In some embodiments, the buffer is sodium phosphate and the nonionic surfactant is 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (TRITON® X. -100). In some embodiments, the buffer is an organic phosphate, optionally sodium phosphate, and the nonionic surfactant is polysorbate 80 (PS80). In some embodiments, the basic amino acid is arginine, the salt is sodium chloride, and the nonionic surfactant is TRITON® X-100. In some embodiments, the nonionic surfactant is TRITON® X-100 and the wash solution also includes histidine. In some embodiments, the nonionic surfactant is TRITON® X-100 and the wash solution also includes histidine and sodium acetate. In some preferred embodiments, the buffer is sodium acetate, the salt is sodium chloride, and the nonionic detergent is TRITON® X-100. In some preferred embodiments, the buffer is sodium acetate, the salt is sodium chloride and the nonionic surfactant is PS80. In some embodiments, the wash liquor has a pH in the range of about 5.0 to about 7.0. In one embodiment, the wash solution has a pH of about 5.0, a pH of about 5.5, a pH of about 6.0, a pH of about 6.5, or a pH of about 7.0. In one embodiment, the wash liquor has a pH of about 6.3.

In some embodiments, the CEX or ProA washes include arginine greater than 0 mM and less than about 75 mM, sodium chloride greater than 0 mM and less than about 30 mM, and anionic surfactant greater than 5 mM and less than about 30 mM. The wash solution comprises about 5 mM to about 10 mM arginine, about 5 mM to about 15 mM arginine, about 5 mM to about 20 mM arginine, about 5 mM to about 25 mM arginine, about 10 mM to about 30 mM arginine, about 10 mM to about 40 mM arginine. , About 10 mM to about 50 mM arginine, about 10 mM to about 60 mM arginine, about 10 mM to about 75 mM arginine, about 20 mM to about 50 mM arginine, or about 20 mM to about 75 mM arginine. In some embodiments, the wash solution comprises about 5 mM arginine, about 10 mM arginine, about 15 mM arginine, about 20 mM arginine, about 25 mM arginine, about 30 mM arginine, about 40 mM arginine, about 50 mM arginine, It contains about 60 mM arginine, or about 75 mM arginine. In some embodiments, the CEX or ProA washes are greater than 0 mM and less than about 25 mM histidine, about 5 mM to about 25 mM histidine, about 5 mM to about 20 mM histidine, about 10 mM to about 25 mM histidine, about 10 mM to about 10 mM. Contains about 20 mM histidine. In some embodiments, the wash solution comprises about 5 mM histidine. In some embodiments, the wash solution comprises about 10 mM histidine, about 15 mM histidine, 20 mM histidine, or about 25 mM histidine.

In some embodiments, the CEX or ProA wash is greater than 0 mM to about 100 mM sodium chloride, about 10 mM to about 25 mM sodium chloride, about 15 mM to about 25 mM sodium chloride, about 25 mM to about 50 mM sodium chloride, It contains about 50 mM to 75 mM sodium chloride, or about 75 mM to about 100 mM sodium chloride. In some embodiments, the wash solution comprises about 10 mM sodium chloride. In some embodiments, the wash solution comprises about 11 mM sodium chloride. In some embodiments, the wash solution is about 5 mM sodium chloride, about 15 mM sodium chloride, about 20 mM sodium chloride, about 25 mM sodium chloride, about 30 mM sodium chloride, about 35 mM sodium chloride, about 40 mM chloride. Sodium, about 45 mM sodium chloride, about 50 mM sodium chloride, about 55 mM sodium chloride, about 60 mM sodium chloride, about 65 mM sodium chloride, about 70 mM sodium chloride, about 75 mM sodium chloride, about 80 mM sodium chloride, Contains about 85 mM sodium chloride, about 90 mM sodium chloride, about 95 mM sodium chloride, or about 100 mM sodium chloride.

In some embodiments, the CEX wash or ProA wash has about 0.00% (w/v) to about 1.0% (w/v) nonionic surfactant, about 0.1% (w/v). v) to about 0.2% (w/v) nonionic surfactant, about 0.05% (w/v) to about 0.15% (w/v) nonionic surfactant, about 0. 0.1% (w/v) to about 0.15% (w/v) nonionic surfactant, about 0.05% (w/v) to about 0.1% (w/v) nonionic Surfactant, or about 0.1% (w/v) to about 0.5% (w/v) nonionic surfactant, or about 0.5% (w/v) to about 1.0% (W/v) nonionic surfactant may be included. In some embodiments, the wash solution comprises about 0.01% (w/v) nonionic surfactant, about 0.05% (w/v) nonionic surfactant, about 0.1% ( w/v) nonionic surfactant, about 0.15% (w/v) nonionic surfactant, about 0.2% (w/v) nonionic surfactant, about 0.25% (W/v) nonionic surfactant, about 0.3% (w/v) nonionic surfactant, about 0.35% (w/v) nonionic surfactant, about 0.4 % (W/v) nonionic surfactant, about 0.45% (w/v) nonionic surfactant, about 5.0% (w/v) nonionic surfactant, about 0. 55% (w/v) nonionic surfactant, about 0.6% (w/v) nonionic surfactant, about 0.65% (w/v) nonionic surfactant, about 0 0.7% (w/v) nonionic surfactant, about 0.75% (w/v) nonionic surfactant, about 0.8% (w/v) nonionic surfactant, about 0.85% (w/v) nonionic surfactant, about 0.9% (w/v) nonionic surfactant, about 0.95% (w/v) nonionic surfactant, Or it contains about 1.0% (w/v) nonionic surfactant. In some embodiments, the nonionic surfactant comprises TRITON® X-100. In some embodiments, the nonionic surfactant comprises PS80. In some embodiments, the nonionic surfactant is lauryl dimethylamine N-oxide (LDAO).

In some embodiments, the CEX wash or ProA wash is greater than 0 mM to about 100 mM sodium acetate, about 5 mM to about 20 mM sodium acetate, about 5 mM to about 30 mM sodium acetate, about 5 mM to about 40 mM sodium acetate, About 5 mM to about 50 mM sodium acetate, about 10 mM to about 25 mM sodium acetate, or about 15 mM to about 30 mM sodium acetate, about 20 mM to about 50 mM sodium acetate, about 25 mM to about 40 mM sodium acetate, about 35 mM to about It contains 50 mM sodium acetate, about 40 to about 65 mM sodium acetate, about 45 mM to about 70 mM sodium acetate, about 65 mM to about 80 mM sodium acetate, about 75 mM to about 100 mM sodium acetate. In some embodiments, the wash solution comprises about 25 mM sodium acetate. In some embodiments, the wash solution is about 5 mM sodium acetate, about 10 mM sodium acetate, about 15 mM sodium acetate, about 20 mM sodium acetate, about 30 mM sodium acetate, about 35 mM sodium acetate, about 40 mM acetic acid. Sodium, about 45 mM sodium acetate, about 50 mM sodium acetate, about 55 mM sodium acetate, about 60 mM sodium acetate, about 65 mM sodium acetate, about 70 mM sodium acetate, about 75 mM sodium acetate, about 80 mM sodium acetate, Contains about 85 mM sodium acetate, about 90 mM sodium acetate, about 95 mM sodium acetate, or about 100 mM sodium acetate.

In some embodiments, the CEX wash or ProA wash contains more than about 0 mM to about 30 mM sodium phosphate. In one preferred embodiment, the wash solution contains about 10 mM sodium phosphate. In some embodiments, the wash solution comprises about 5 mM to about 10 mM sodium phosphate, about 5 mM to about 15 mM sodium phosphate, about 5 mM to 20 mM sodium phosphate, about 10 mM to 25 mM sodium phosphate, or about. Contains 10 mM to 30 mM sodium phosphate. In some embodiments, the wash solution is about 5 mM sodium phosphate, about 10 mM sodium phosphate, about 15 mM sodium phosphate, about 20 mM sodium phosphate, about 25 mM sodium phosphate or about 30 mM phosphate. Contains sodium.

In some embodiments, the CEX or ProA washes include greater than about 0 mM to about 100 mM sodium phosphate, and greater than 0.0% to about 1.0% nonionic surfactant. In some embodiments, the wash solution is greater than 0 mM to about 100 mM sodium acetate, greater than 0 mM to about 100 mM sodium chloride, and greater than 0.0% to about 0 mM and less than about 500 mM arginine, greater than 0 mM to about 100 mM. Includes sodium chloride, greater than 0 mM and less than about 25 mM histidine, greater than 0 mM to about 50 mM sodium acetate, and greater than 0.0% to about 1.0% nonionic surfactant.

Any of the wash solutions of the present invention can be used to purify the protein of interest using a CEX chromatography support.

In certain embodiments, 1 to 10 column volumes (CV) of wash solution are applied to the CEX chromatography support. In certain embodiments, 1 to 9 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 1 to 8 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 1 to 7 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 1 to 6 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 1 to 5 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 1 to 4 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 1 to 3 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 1 to 2 CV of wash solution is applied to the CEX chromatography support.

In certain embodiments, 1 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 2 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 3 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 4 CV of wash solution is applied to the CEX chromatography support. In certain embodiments, 5 CV of wash solution is applied to the CEX chromatography support.

In certain embodiments of the methods provided herein, washing with a detergent-containing solution includes, for example, washing with a detergent-free solution prior to eluting the protein of interest. Can be followed later. Washing with a detergent-free solution can remove the detergent. Washing with a detergent-removing solution increases the yield, for example, by eliminating the need to discard this first fraction because the first fraction of the elution contains detergent. Has been shown to improve the HCP exclusion performance of CEX chromatography.

In certain embodiments, 1 to 10 column volumes (CV) of wash solution without detergent are applied to the CEX chromatography support. In certain embodiments, 1 to 9 CV of detergent-free wash is applied to the CEX chromatography support. In certain embodiments, 1-8 CV of detergent-free wash is applied to the CEX chromatography support. In certain embodiments, 1 to 7 CV of detergent-free wash is applied to the CEX chromatography support. In certain embodiments, 1 to 6 CV of detergent-free wash is applied to the CEX chromatography support. In certain embodiments, 1 to 5 CV of detergent-free wash is applied to the CEX chromatography support. In certain embodiments, 1 to 4 CV detergent-free wash is applied to the CEX chromatography support. In certain embodiments, 1 to 3 CV of detergent-free wash is applied to the CEX chromatography support. In certain embodiments, 1 to 2 CV of detergent-free wash is applied to the CEX chromatography support.

In certain embodiments, 1 CV of detergent-free wash is applied to the CEX chromatography support. In certain embodiments, a detergent-free 2 CV wash is applied to the CEX chromatography support. In certain embodiments, a detergent-free 3 CV wash is applied to the CEX chromatography support. In certain embodiments, a detergent-free 4 CV wash is applied to the CEX chromatography support. In certain embodiments, a detergent-free 5 CV wash is applied to the CEX chromatography support.

In certain embodiments of the methods provided herein, washing with a solution containing detergent elutes the protein of interest without washing with a solution containing no detergent prior to elution. It doesn't come after that. In embodiments such as these, it is possible to discard the first fraction of the elution, which may contain detergent.

Embodiments of the present disclosure describe in detail the following non-limiting examples that describe in detail the preparation of the wash liquor of the present disclosure and the method for using the wash liquor of the present disclosure for purification of a protein of interest. It can be further defined by reference. It will be apparent to those skilled in the art that many modifications to both materials and methods can be made without departing from the scope of this disclosure.

The examples and embodiments described herein are for illustration purposes only, and various modifications or alterations in light thereof will be suggested to those skilled in the art and should be included within the spirit and scope of the present application. It is understood that

Example 1-Protein A Wash Fluid Materials and Methods for Virus Clearance Materials and Equipment Chinese hamster ovary (CHO) cells were used to express the monoclonal antibody used in these examples. MABSELECT SURE® Protein A resin was purchased from GE Healthcare (Uppsala, Sweden). All buffers were prepared using ultrapure water obtained from the Millipore water purification system. Buffers and chemicals used for solution preparation were obtained from JT Baker (Philipsburg, NJ). All chromatography experiments were performed on an Akta Avant liquid chromatography system (GE Healthcare Life Sciences, Marlborough, MA). This equipment includes an integrated UV and conductivity monitoring system. Unicorn software (GE Healthcare Life Sciences, Marlborough, MA) was used for system control and sampling.

Protein A chromatography. The MABSELECT SURE® chromatography column was equilibrated with 1×PBS against 5 CV (column volume). After equilibration, harvested cell culture fluid (HCCF) was loaded onto the column at a loading volume of 40 grams of mAb per liter of resin. After loading application, the column was first washed with 3 CV of 1×PBS buffer followed by a second wash with 5 CV of candidate wash buffer. The column was subsequently washed with a third wash using 5 CV of 5 mM succinic acid, pH 5.8 buffer. The mAb was eluted from the column using 5 CV of 25 mM glycine, 10 mM succinic acid, pH 3.7 buffer. A wash in place was applied after production.

Quantitative ELISA-Host cell derived protein (HCP). Host Cell Derived Protein (HCP) was determined by the CHO Host Cell Derived Protein Third Generation Kit (Immunoenzymetric Assay for the Measurement of CHO Host Cell Proteins, Catalog # F550, Cygnus Technologies, Co., Ltd.) according to the manufacturer's protocol. .. Absorbance data at 450/650 nm was acquired on a SPECTRAMAX® Plus microplate reader (Molecular Devices, Sunnyvale CA) and by SOFTMAX® Pro 6.4.2 software (Molecular Devices, Sunnyvale, CA). Was analyzed. The HCP value was calculated from the logistic goodness of fit of the four parameters of the standard curve prepared from the standard material contained in the CHO host cell-derived protein third generation kit.

Viral Exclusion Results Seven selected washes were studied for RVLP exclusion during Protein A chromatography washes and the results are summarized in Table 1. TRITON® X-100 was very effective at in vitro RVLP exclusion.

In addition, virus clearance from selected lavage fluids was evaluated by using a spike-in study by using two model viruses: xenotropic murine leukemia virus (X-MulV) and mouse microvirus (MVM). The compositions and results are shown in Table 2. These results show a 1.7-log (arginine) or 0.8-log (arginine + guanidine) improvement in X-MulV exclusion compared to a control wash of 5 mM succinic acid at pH 5.8, and It is shown that there was a 1.3-log improvement in MVM exclusion by the wash containing either arginine- or arginine + guanidine.

Example 2-Wash Solution for Cation Exchange Chromatography Materials and Methods Polymeric ion exchange chromatography resin (Poros™ XS, Life Technologies Corp., Carlsbad, CA), and strong anion exchange of quaternary ammonium (Q). A resin composed of 6% agarose beads cross-linked by a group (Q Sepharose® Fast Flow, GE-Healthcare Life Sciences, Marlborough, MA) was utilized for CEX chromatography. All buffers used in this study were prepared using ultrapure water obtained from a water purification system (EMD Millipore, Billerica MA). Buffers and chemicals used for solution preparation were obtained from JT Baker (Philipsburg, NJ). All chromatography experiments were performed on an Akta Avant chromatography system (GE Healthcare Life Sciences, Marlborough, MA).

The cation exchange chromatography column was equilibrated with equilibration buffer of pH 5.5 for 5 CV (column volume), low conductivity (<5 mS/cm). After equilibration, the pH-adjusted Protein A purified product pool (pH 5.5) was loaded onto the column at a loading volume of 50-80 grams of mAb per liter of resin. After loading application, the column was first washed with 3 CV of EQ buffer, followed by wash 2 with 5 CV of candidate wash buffer. The column was then washed with Wash 3 using 5 CV of 10 mM phosphate, pH 6.0 buffer. The mAb was eluted from the column using 5 CV of 20 mM phosphate, pH 7.0 buffer. A wash in place was applied after production.

Quantitative ELISA-Host Cell Derived Protein (HCP)
Host cell-derived protein (HCP) was determined by the CHO Host Cell Proteins 3rd Generation kit (Immunoenzymetric Assay for the Measurement of CHO Host Cell Proteins, Catalog, Facts, Catalog #F5) according to the manufacturer's protocol. Absorbance data at 450/650 nm was acquired on a SpectraMax Plus microplate reader and analyzed by SoftMaxPro 6.4.2 software (Molecular Devices, Sunnyvale, CA). HCP values were calculated from the four parameter logistic fit of a standard curve made from standards included in the CHO Host Cell Proteins 3rd Generation kit.

HPLC-SEC
The purity of the product sample was determined by HPLC-using a Waters HPLC system (2695 separation module and 2996 photodiode array detector) on a TSK gel G3000SW column (7.5 mm ID x 30 cm, 10 μm average particle size, Tosoh Bioscience, Japan). Analyzed by SEC method. PBS buffer pH 6.8 was used as mobile phase at a flow rate of 1 ml/min. Injection volume was 100-125 μg protein.

Results and Discussion The protein of interest used in this study was an anti-TL1a antibody containing a heavy chain variable region having the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6 and a light chain variable region having the amino acid sequence of SEQ ID NO:7. Met. A wash liquor containing a total of four detergents was investigated in this study. This study was essentially repeated with anti-CGRP antibody (V _H of SEQ ID NO:1, V _{L of} SEQ ID NO:2) and anti-CD38 antibody (V _H of SEQ ID NO:3, V _{L of} SEQ ID NO:4). The formulation table of the buffer solution of Wash 2 and the results are shown in Tables 3 to 8.

All four detergent-containing washes achieved similar impurity exclusion for anti-TL1a, anti-CGRP, and anti-CD38 antibody molecules, as compared to control conditions that contained no detergent. No residual surfactant was observed in the elution pool. A detergent wash can be used to improve virus clearance while maintaining the quality of the purified mAb.

This study demonstrated that detergent cleaning does not reduce product quality. Both residual detergent and host cell derived protein (HCP) can be eliminated from the product using detergent washes.

Example 3-Cation Exchange Materials and Methods as Polishing and Capture Columns for Virus Exclusion After first equilibrating the cation exchange chromatography column, the packing material was loaded with a resin loading volume of 40-100 mg/ml. Packed on a column. After washing the column, the elution buffer was used to elute the monoclonal antibody (mAb) product from the column. In-place wash (CIP) and in-place sanitization (SIP) procedures were applied after product elution.

The antibody titer was determined on a Waters Alliance HPLC system using HPLC Protein A titer 2.1 mm x 30 mm analytical affinity column (Poros A affinity column, ABI.P/N: 2-1001-00) and appropriate standards. Was quantified by. The standard content ranged from 1 to 100 μg in a 10 μL injection volume. Standards and samples were injected at 10 μL. Mobile phase A was 1X PBS buffer (ThermoFisher Scientific, Cat. #: 10010072). Mobile phase B was 12 mM HCl. The flow rate was 2.5 mg/mL. The antibody was eluted from the column with a gradient of 0-70% mobile phase B for 2.5 minutes. The elution peak was integrated. The areas of the elution peaks were plotted against these areas and a linear standard curve derived from the content of standards to determine antibody titers.

Solo VPE Protein Concentration Measurement Protein concentration was measured on a Solo VPE system (C Technologies Inc.). For A280 values, typically 50-200 μL of sample was added in a small silica container. The protein concentration was calculated by the Solo VPE system based on the molecular extinction coefficient.

HPLC-size exclusion chromatography (SEC)
The antibody purity monomer percentage (%) was preceded by a 6.0 mm x 4.0 cm guard column (TOSOH TSKgel Guard SWxl, P/N: 08543) size exclusion column (TSKgel G3000SWxl, P). /N:08541) by size exclusion chromatography on a Waters Alliance HPLC system. A typical sample was injected at 5.0 mg/mL in 30 μL. The isocratic mobile phase was 100 mM sodium phosphate, 250 mM sodium chloride, pH 6.5. The flow rate was 0.5 mg/mL. High molecular weight species, dimers, monomers, and small molecular weight species were separated in 35 minutes. The various percentages were determined by the peak area divided by the total area of all peaks.

Quantitative ELISA-Host Cell Derived Protein (HCP)
HCP levels in the samples were determined by an ELISA assay using the Cygnus CHO HCP ELISA kit (Cygnus technologies, Cat. #: F550) according to the manufacturer's protocol.

X-MuLV Spike-In Study The CEX conditions described above in this example were carried out using a packing material supplemented with X-Mulv virus. Viral content in loaded and eluted samples was analyzed by a cell-based infection assay, TCID50. The LRV of each run was calculated by subtracting the log10 total PFU of the eluted sample from the log10 total PFU of the loaded sample.

RVLP qPCR Assay RVLPs (retrovirus-like particles) were quantified by qPCR. All nucleic acids were isolated from the samples using the MagMAX™ viral RNA isolation kit (ThermoFisher, Cat #AM1939) according to the manufacturer's protocol. All nucleic acids were first treated with esDNase (to remove genomic DNA) and then using SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme kit (ThermoFisher, Cat #AM1939). And reverse transcribed into cDNA. The cDNA sample underwent Taqman-based qPCR targeting the 3027 to 3125 bp region of the RVLP gene (GeneBank Accession #: U09104.1). The primers used for qPCR were CCTGAGTCACCGGACTCGCAT (SEQ ID NO:8) and ACCAGTCGCGAGGCTGGAG (SEQ ID NO:9). They were purchased from Integrated DNA Technologies (Skokie, Illinois). The probe used for qPCR was 5-FAM-AGGGAGGCTACAGGCGG-MGBNFQ-3 (SEQ ID NO: 10) purchased from ThermoFisher. The standard for qPCR was a double-stranded DNA fragment covering the amplicon region. The qPCR assay was performed on a 7500 FAST system (ThermoFisher) using the Taqman Fast Universal PCR master mix (ThermoFisher, Cat#4352046).

Residual Triton-100
The concentration of residual Triton-100 was quantified by HPLC on a Waters Alliance HPLC system using a 4.6 mm x 150 mm XDB-C18 column (Agilent Technologies, P/N: 993967-902). The sample was mixed with an equal volume of 100% methanol and centrifuged at 25,000 xg for 30 minutes at 4°C. The sample supernatant was saved for HPLC injection. Triton X-100 standards were diluted in 50% methanol prior to HPLC injection. Triton X-100 was eluted using a gradient of 78% to 100% methanol. The Triton X-100 peak was integrated. The concentration of Triton X-100 in the samples was calculated by plotting the corresponding peak areas against these standards.

CEX as a polishing column
To assess the effectiveness of CEX as a polishing column, two assays were performed using different antibodies, resin, equilibration buffer, and wash conditions in the presence or absence of detergent.

In the first assay, the process conditions listed in Table 10 were applied on SP Sepharose Fast Flow (SPFF) resin to purify anti-CGRP IgG2 antibodies. The resin is a strong cation exchange chromatography of Sepharose-based sulfopropyl produced by GE. Similar resins include Fractogel EMD SO3-, and Capto S.

A virus spike-in study was performed using a virus loaded packing material. The conditions for Wash 2 tested and their results are shown in Table 11.

These results demonstrate that the addition of 0.5% Triton in Wash 2 significantly improves the log clearance of X-MuLV.

The product quality was also evaluated using the Protein A purified mAb as packing material under the conditions summarized in Table 12, and these results are also reported in this table.

These results in Table 12 demonstrate that the addition of 0.5% Triton in Wash 2 did not reduce product quality in terms of either% monomer or residual host cell derived protein (HCP). .. Also, these results demonstrate that Wash 3 is capable of removing residual surfactant to undetectable levels.

In the second assay, the process conditions listed in Table 13 were applied on Poros XS resin (Thermo Scientific) to purify anti-TL1a IgG1 antibodies. This resin cross-linked a poly(styrenedivinylbenzene) polymer matrix with a sulfopropyl ligand.

A virus spike-in study was performed using a virus loaded packing material. The conditions for Wash 2 tested and their results are shown in Table 14.

These results demonstrate that for Assay 1, the addition of 0.5% Triton in Wash 2 significantly improves the log clearance of X-MuLV in Assay 2. In addition, it was possible to achieve >6 logs of viral clearance in 5 column volume (CV) washes with or without a 30 minute pause at the end of Wash 2.

We also evaluated the product quality using Protein A purified mAb as packing material under the conditions summarized in Table 15, and these results are also reported in this table.

These results in Table 15 show that for assay 1, the addition of 0.5% Triton in wash 2 was either product percent in terms of% monomer or residual host cell derived protein (HCP) in assay 2. Demonstrate that it does not reduce. However, the residual surfactant was not efficiently removed without Wash 3 probably because Poros XS is a hydrophobic resin. In all cases, the addition of Wash 3 lowered the surfactant levels below the limit of detection.

CEX as a capture column
To assess the effectiveness of the effect of detergents on CEX as a capture column, different resins, equilibration buffers, and wash conditions were used in the presence or absence of detergents to Two assays were performed.

In the first assay, the process conditions listed in Table 16 were applied on SPFF resin.

Retrovirus-like particle (RVLP) exclusion was evaluated using HCCF as the packing material and these conditions are listed in Table 17.

These results demonstrate that the addition of 0.5% Triton in Wash 2 improved RVLP exclusion by 2 logs and also significantly increased HCP exclusion. The volume of Wash 2 did not affect the effectiveness of virus clearance and 1 column volume (CV) was sufficient for Wash 2. However, increasing the volume of Wash 2 did not improve HCP exclusion. Also, having Wash 3 improved HCP exclusion.

Additional tests were performed with modified Wash 2 conditions, as summarized in Table 18. In these tests, 5 column volumes (CV) of Wash 2 and 5 CV of Wash 3 were applied. In addition, these results are reported in Table 17.

These results in Table 18 show that 0.05% Triton, >0.2% SB-12, >0.2% SB-14, >0.5% NP-40 and >0.05. Demonstrate that the addition of% LDAO was all effective for RVLP exclusion. They were able to eliminate RVLP until subsequent detection. Moreover, LDAO, SB-12, and SB-14 can significantly improve HCP exclusion.

In the second assay, the process conditions listed in Table 19 were applied on Poros XS resin.

Retrovirus-like particle (RVLP) exclusion was evaluated using HCCF as the packing material and these conditions are listed in Table 20. These results are also shown in this table.

These results demonstrate that HCP with Wash 3 is significantly lower than without Wash 3.

Additional tests were performed with modified Wash 2 conditions, as summarized in Table 21. In these tests, 5 column volumes (CV) of Wash 2 and 5 CV of Wash 3 were applied. In addition, these results are reported in Table 21.

These results in Table 21 demonstrate that a concentration of Triton of at least 0.1% is effective in virus clearance.

In summary, these results, described in Example 3 herein, show that the interface to the CEX cleaning (wash 2), whether CEX is used in the polishing step or CEX in the capture step, is We demonstrate that the addition of active agents improves retroviral clearance. The addition of surfactant does not adversely affect the process yield or product quality in terms of either HCP or% monomer purity. This detergent-based virus clearance can be achieved on Sepharose matrix resins and synthetic polymer matrix resins, and one column volume is sufficient to achieve effective RVLP exclusion. In addition, wash 3 improves HCP exclusion, and LDAO and 0.5% Triton improve HCP exclusion on Sepharose-based resins, respectively.

The present invention is not limited to the embodiments described and illustrated above, but is capable of variation and modification within the scope of the appended claims.

Throughout this specification various publications, including patents, patent applications, accession numbers, technical and academic papers, are cited. Each of these cited publications is incorporated herein by reference in its entirety and for all purposes.
Sequence Listing Anti-CGRP VH (SEQ ID NO: 1)
1 EVQLVESGGG LVQPGGSLRL SCAASGFTFFS NYWISWVRQA PGKGLEWVAE 50
51 IRSESDASAT HYAEAVKGRF TISRDNAKNS LYLQMNSLRA EDTAVYYCLA 100
101 YFDYGLAIQN YWGQGTLVTV SS
Anti-CGRP VL (SEQ ID NO: 2)
1 EIVLTQSPAT LSSPGERAT LSCKASKRVT TYVSWYQQKP GQAPRLLIYG 50
51 ASNRYLGIPA RFSGGSGSGT FTLITISLEP EDFAVYCSQ SYNYPYTFGQ 100
101 GTKLEIK
Anti-CD38 VH (SEQ ID NO:3)
1 EVQLVQSGAE VKKPGATVKI SCKVSGYTFT DSVMNWVQQA PGKGLEWMGW 50
51 IDPEYGRTDV AEKFQGRVTI TADTSTDTDTAY MELSSLRSED TAVYYCARTK 100
101 YNSGYGFPYW GQGTTVTVSS
Anti-CD38 VL (SEQ ID NO:4)
1 DIQMTQSPSS LSASVGDRVT ITCKASQNVD SDVDWYQQKP GKAPKLLIYK 50
51 ASNDYTGVPS RFSGSGSGGT FTTIISSPQP EDIATYYCMQ SNTHPRTFGG 100
101 GTKVEIKR
Anti-TL1a VH1 (SEQ ID NO:5)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWLNPNSGNGTY
AQKFQGRVTMTADRSTSTAYMELSSLRSEDTAVYYCAREVPETAAFEYWGQGTLVTVSS
Anti-TL1a VH2 (SEQ ID NO:6)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQAPGQGLEWMGWLNPNSGYTGY
AQKFQGRVTMTADRSTSTAYMELSSLRSEDTAVYYCAREVPETAAFEYWGQGTLVTVSS
Anti-TL1a VL (SEQ ID NO:7)
QSVLTQPPSVSGAPGQRVTISCTSSSS DIGAXXGVXWYQQLPGTAPKLLIEGYYNRPSGVPDRFSGSGSKTSTASLTITGLLPEDEGDYYQQSXDGTLSALFGGGGTVLTVLG
Xaa32 is G or A.
Xaa33 is L or S or Q.
Xaa36 is H or L.
Xaa93 is Y or F or W.

Claims (113)

A method of purifying a protein of interest bound to a CEX chromatography support comprising applying a wash solution containing one or more detergents to a cation exchange (CEX) chromatography support. 2. The method of claim 1, further comprising eluting the protein of interest from the support to form a purified eluate containing the protein of interest. A CEX chromatography support is loaded with a mixture containing the protein of interest and one or more contaminating proteins, aggregates, and/or viruses, and the support is washed with a wash solution containing one or more detergents. And then forming the purified eluate of the protein of interest by eluting the protein of interest from the support. A CEX chromatography support is loaded with a mixture containing the protein of interest and one or more contaminating proteins, aggregates, and/or viruses, and the support is washed with an aqueous wash containing one or more surfactants. After washing to elute the one or more contaminating proteins, aggregates, and/or viruses from the support, the protein of interest is eluted from the support to remove the protein of interest. A method for purifying the protein of interest, comprising forming a purified eluate. The one or more surfactants include lauryl dimethylamine N-oxide (LDAO), Triton 100, nonylphenoxy polyethoxyl ethanol (NP40), sulfobetaine-12 (SB-12), sulfobetaine-14 (SB-14). 5. The method of any one of claims 1 to 4, comprising: Polysorbate 20 (PS20), Polysorbate 80 (PS80), or a combination thereof. 6. The method of any one of claims 1-5, wherein the one or more surfactants comprises a zwitterionic surfactant. The zwitterionic surfactant is 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), or cocamidopropyl hydroxysultaine (CAHS), or a combination thereof. 7. The method according to claim 6. 8. The method of any one of claims 1-7, wherein the one or more surfactants comprises a quaternary ammonium salt. 9. The method of claim 8, wherein the quaternary ammonium salt is cetrimonium bromide (CTAB) or dioctadecyldimethyl ammonium bromide (DODAB). 10. The method of any one of claims 1-9, wherein the one or more surfactants comprises an alkylphenol ethoxylate. 11. The method of claim 10, wherein the alkylphenol ethoxylate is nonoxynol, optionally nonaethylene glycol. 12. The method of any one of claims 1-11, wherein the one or more surfactants comprises ethoxylates. 13. The method of any one of claims 1-12, wherein the one or more surfactants comprises an alkyl polyglucoside, optionally decyl glucoside. 14. The method of any one of claims 1-13, wherein the one or more surfactants comprises an amino oxide. 15. The method of any one of claims 1-14, wherein the one or more surfactants comprises phosphine oxide. 16. The method of any one of claims 1-15, wherein the one or more surfactants comprises a nonionic surfactant. 17. The method of any one of claims 1-16, wherein the one or more surfactants comprises an anionic surfactant and optionally the anionic surfactant is sodium dodecyl sulfate (SDS). .. 18. The method of any one of claims 1-17, wherein the wash liquor comprises from about 0.01 w/v% to about 1 w/v% of the one or more surfactants. 19. The method of claim 18, wherein the wash solution comprises about 0.05 w/v% to about 0.5 w/v% of the one or more surfactants. 19. The method of claim 18, wherein the wash solution comprises about 0.1 w/v% of the one or more surfactants. 19. The method of claim 18, wherein the wash solution comprises about 0.2 w/v% of the one or more surfactants. 19. The method of claim 18, wherein the wash solution comprises about 0.5 w/v% of the one or more surfactants. 23. The method of any one of claims 1-22, wherein the CEX chromatography support is a Sepharose matrix resin. 23. The method of any one of claims 1-22, wherein the CEX chromatography support is a synthetic polymer matrix resin. 23. The method of any one of claims 1-22, wherein the CEX chromatography support is not a hydrophilic resin. The CEX chromatography support may be Capto S, Fractogel EMD SO3-, Fractogel EMD TMAE, Fractogel EMD, Eshnumo S, Eshmuno HCX, Tohso Toyopar, and Tosoh Toyopur, and Tosoh Topo, Tosoh and Tosoh. 23. The method according to any one of 1 to 22. 27. The method of any one of claims 2 to 26, further comprising applying a subsequent wash solution to the CEX chromatography support prior to eluting the protein of interest from the support. 28. The method of claim 27, wherein the subsequent wash solution is free of surfactant. 29. The method of claim 28, wherein the subsequent wash liquid removes the surfactant from the support. 30. The method of any one of claims 27-29, wherein applying the subsequent wash solution increases host cell derived protein (HCP) removal compared to not applying the subsequent wash solution. 6. The method of claim 1, 2, and 5, further comprising loading harvested cell culture fluid (HCCF) onto the support prior to applying the wash solution containing the one or more surfactants to the support. 31. The method according to any one of items 1 to 30. 31. A method according to any one of claims 3 to 30, wherein the mixture comprising the protein of interest and one or more contaminating proteins, aggregates and/or viruses is HCCF. 33. The method of any one of claims 1-32, wherein the method does not include Protein A purification. 6. The method of claim 1, 2, and 5, further comprising loading the eluate of a chromatography step onto the support prior to applying the wash solution containing the one or more surfactants to the support. 31. The method according to any one of items 1 to 30. 31. A method according to any one of claims 3 to 30, wherein the mixture comprising the protein of interest and one or more contaminating proteins, aggregates and/or viruses is the eluate of a chromatography step. 36. The method according to claim 34 or 35, wherein the eluent of the chromatography step is an eluent of an affinity chromatography step. 37. The method of claim 36, wherein the affinity chromatography step is Protein A purification. 38. The method of claim 37, wherein the Protein A purification comprises washing the Protein A support with a wash solution containing one or more detergents. 38. The method of claim 37, wherein the Protein A purification does not include washing the Protein A support with a wash solution containing one or more detergents. 40. The method of any one of claims 1-39, wherein the wash liquor excludes viruses as measured by LRV. a) loading a mixture containing the protein of interest and one or more contaminants, aggregates and/or viruses on a Protein A chromatography support,
b) washing the support with a Protein A chromatography wash containing one or more detergents to remove the one or more contaminating proteins, aggregates and/or viruses from the Protein A chromatography support. ,
c) eluting the protein of interest from the Protein A chromatography support,
d) loading the eluate containing the protein of interest on a cation exchange (CEX) chromatography support,
e) washing the CEX chromatography support with a CEX chromatography wash containing one or more detergents to remove the one or more contaminating proteins, aggregates and/or viruses from the support, And f) eluting the protein of interest from the CEX chromatography support to form a purified eluate of the protein of interest,
A method for purifying a protein, comprising: 42. The method of any one of claims 3 to 30, 32, 33, and 35 to 41, wherein the contaminating protein is a host cell derived protein. 43. The method of claim 42, wherein the host cell-derived protein is derived from Chinese hamster ovary cells. 44. The method of any one of claims 41-43, wherein the Protein A wash eliminates virus as measured by log reduction value (LRV). 45. The method of any one of claims 41-44, wherein the CEX wash solution excludes viruses as measured by LRV. The LRV is between about 1.0 to about 10.0Log _10, The method according to any one of claims 40, 44, and 45. The LRV is between about 1.0 to about 5.0 log _10, The method of claim 46. 47. The method of claim 46, wherein the LRV is between about 4.0 and about 8.0 log ₁₀ . The LRV is about 3.0 log _10, The method of claim 46. The LRV is about 4.0Log _10, The method of claim 46. The LRV is about 5.0 log _10, The method of claim 46. The LRV is about 6.0 log _10, The method of claim 46. 53. The method of any one of claims 40 and 44-52, wherein the LRV is measured by quantitative PCR. 53. The method of any one of claims 40 and 44-52, wherein the LRV is measured using an infection assay. 55. The method of any one of claims 40 and 44-54, wherein the LRV is achieved by applying one column volume (CV) of the wash. 55. The method of any one of claims 40 and 44-54, wherein the LRV is achieved by applying 2 CVs of the wash. 55. The method of any one of claims 40 and 44-54, wherein the LRV is achieved by applying 3 CVs of the wash. 55. The method of any one of claims 40 and 44-54, wherein the LRV is achieved by applying 4 CVs of the wash. 55. The method of any one of claims 40 and 44-54, wherein the LRV is achieved by applying 5 CV of the wash. 60. Any of claims 1-59, further comprising applying a detergent-free wash solution to the support prior to applying the wash solution containing the one or more surfactants to the support. The method described in the section. 61. The method of any one of claims 2-60, further comprising treating the purified eluate of the protein of interest by diafiltration, ultrafiltration, or both diafiltration and ultrafiltration. the method of. Further comprising loading the purified eluate of the protein of interest on a membrane chromatography support, and collecting a flow-through containing the further purified eluate from the membrane chromatography support. The method according to any one of claims 2 to 60. 63. The method of any one of claims 1-62, wherein the method does not include an anion exchange chromatography step. 64. The method of any one of claims 2 to 63, further comprising subjecting the eluate to a virus inactivation step. 65. The method of claim 64, wherein said virus inactivation is achieved by low pH virus inactivation to form a virus inactivated preparation. 66. The method of claim 65, wherein the pH drops between about 2.5 and about 5. 67. The method of claim 65 or 66, further comprising lowering the pH of the purified eluate of the protein of interest for a period of time sufficient to inactivate the virus. 68. The method of any one of claims 2 to 67, further comprising filtering the purified eluate of the protein of interest to remove virus. 69. The method of any one of claims 2 to 68, further comprising formulating the purified eluate of the protein of interest as a composition comprising a pharmaceutically acceptable excipient. .. 70. The method of any one of claims 1-69, further comprising expressing the protein of interest and one or more contaminating proteins in a bioreactor having a capacity of at least about 250 liters. 71. The method of claim 70, wherein the bioreactor has a capacity of at least about 500 liters, at least about 2000 liters, or at least about 5000 liters. 72. The method of any one of claims 1-71, wherein the protein of interest comprises an antibody or antigen binding fragment thereof, or a fusion protein construct thereof. The antibody or antigen-binding fragment thereof,
(A) optionally said antibody or antigen binding fragment thereof comprises a VH having the amino acid sequence of SEQ ID NO:5 or 6, and a VL having the amino acid sequence of SEQ ID NO:7, specifically for TNF-like ligand 1A (TL1a) Join,
(B) optionally said antibody or antigen-binding fragment thereof comprises VH having the amino acid sequence of SEQ ID NO: 1 and VL having the amino acid sequence of SEQ ID NO: 2 and specifically binds to calcitonin gene-related peptide (CGRP) Or (c) optionally said antibody or antigen-binding fragment thereof comprises VH having the amino acid sequence of SEQ ID NO:3 and VL having the amino acid sequence of SEQ ID NO:4 and specifically binds to CD38,
73. The method of claim 72. The wash solution comprises one or more detergents, the use of said wash solution in CEX chromatography to purify a protein of interest bound to a cation exchange (CEX) support. A CEX chromatography wash for purifying a protein of interest bound to a cation exchange (CEX) support, which comprises a detergent. 76. The protein of any one of claims 46-75, wherein the Protein A chromatography wash or the CEX chromatography wash comprises about 0.1 w/v% to about 0.6 w/v% of the detergent. Use, method or cleaning solution. 77. The protein of any one of claims 46-76, wherein the Protein A chromatography wash or the CEX chromatography wash comprises about 0.2 w/v% to about 0.5 w/v% of the detergent. Use, method or cleaning solution. 78. The use, method or wash solution of any one of claims 46-77, wherein the Protein A chromatography wash solution or the CEX chromatography wash solution comprises about 0.2 w/v% of the detergent. 78. The use, method or wash solution of any one of claims 46-77, wherein the Protein A chromatography wash solution or the CEX chromatography wash solution contains about 0.3 w/v% of the detergent. 78. The use, method or wash solution of any one of claims 46-77, wherein the Protein A chromatography wash solution or the CEX chromatography wash solution contains about 0.5 w/v% of the detergent. 77. The use, method or wash solution of any one of claims 46-76, wherein the Protein A chromatography wash solution or the CEX chromatography wash solution contains about 0.6 w/v% of the detergent. 82. The use, method or cleaning solution according to any one of claims 46 to 81, wherein the surfactant is a nonionic surfactant. 83. The use, method or wash solution of any one of claims 46-82, wherein the surfactant is TRITON(R) X-100. 84. The use, method or wash solution of claim 83, comprising about 0.2% TRITON ® X-100. 84. The use, method or wash solution of claim 83, which comprises about 0.5% TRITON® X-100. 83. The use, method or wash solution of any one of claims 46-82, wherein the surfactant is PS80. 87. The use, method or wash solution of claim 86, comprising about 0.3% PS80. 87. The use, method or wash solution of claim 86, comprising about 0.6% PS80. 89. Any of claims 1-88, wherein said Protein A chromatography wash or said CEX chromatography wash further comprises arginine, optionally said Protein A chromatography wash or said CEX chromatography wash comprises about 25 mM arginine. The use, method or cleaning solution as described in 1 above. 89. Any of claims 1-89, wherein said Protein A chromatography wash or said CEX chromatography wash further comprises histidine, and optionally said protein A chromatography wash or said CEX chromatography wash comprises about 5 mM histidine. The use, method or cleaning solution as described in 1 above. The protein A chromatography wash or the CEX chromatography wash optionally further comprises sodium chloride, and the protein A chromatography wash or the CEX chromatography wash comprises about 5 mM to about 15 mM sodium chloride. Use, method or cleaning liquid according to any one of 1 to 90. 92. The use, method or wash solution of claim 91, wherein the Protein A chromatography wash solution or the CEX chromatography wash solution comprises about 10 mM sodium chloride, or about 11 mM sodium chloride. The protein A chromatography wash or the CEX chromatography wash further comprises sodium phosphate, optionally the protein A chromatography wash or the CEX chromatography wash comprises about 10 mM sodium phosphate. 93. The use, method or cleaning solution according to any one of 1 to 92. The protein A chromatography wash or the CEX chromatography wash further comprises sodium acetate, optionally the protein A chromatography wash or the CEX chromatography wash comprises about 20 mM to about 50 mM sodium acetate. The use, method or cleaning solution according to any one of 1 to 93. 95. The use, method or wash solution of any one of claims 93 or 94, wherein the Protein A chromatography wash solution or the CEX chromatography wash solution comprises about 25 mM sodium acetate, or about 50 mM sodium acetate. The protein A chromatography washing solution or the CEX chromatography washing solution is
(A) about 10 mM sodium phosphate and about 0.2% TRITON® X-100,
(B) about 10 mM sodium phosphate and about 0.5% TRITON® X-100,
(C) about 10 mM sodium phosphate and about 0.3% PS80, or (d) about 10 mM sodium phosphate and about 0.6% PS80,
Use, method or cleaning liquid according to any one of claims 1 to 4 and 23 to 75, comprising. The protein A chromatography washing solution or the CEX chromatography washing solution is
(A) about 50 mM sodium acetate, about 25 mM arginine, about 5 mM histidine, about 11 mM sodium chloride and about 0.3% PS80,
(B) about 50 mM sodium acetate, about 25 mM arginine, about 5 mM histidine, about 11 mM sodium chloride and about 0.6% PS80,
(C) about 50 mM sodium acetate, about 25 mM arginine, about 5 mM histidine, about 11 mM sodium chloride and about 0.2% TRITON® X-100,
(D) about 50 mM sodium acetate, about 25 mM arginine, about 5 mM histidine, about 11 mM sodium chloride and about 0.5% TRITON® X-100,
(E) about 25 mM sodium acetate, about 10 mM sodium chloride and about 0.3% PS80,
(F) about 25 mM sodium acetate, about 10 mM sodium chloride and about 0.6% PS80,
(G) about 25 mM sodium acetate, about 10 mM sodium chloride and about 0.2% TRITON® X-100, or (h) about 25 mM sodium acetate, about 10 mM sodium chloride and about 0.5. % TRITON® X-100,
Use, method or cleaning liquid according to any one of claims 1 to 4 and 23 to 75, comprising. 98. The use, method or wash solution of any one of claims 1-97, wherein the Protein A chromatography wash solution or the CEX chromatography wash solution has a pH of about 5.0 to 7.0. 99. The use, method or wash solution of claim 98, wherein the Protein A chromatography wash solution or the CEX chromatography wash solution has a pH of about 5.5 or about 6.3. 102. A preparation of a protein of interest produced by the method of any one of claims 1-73 and 76-99. 98. A virus inactivated preparation of a protein of interest produced by the method of any one of claims 1-73 and 76-99. 102. The preparation of claim 100 or 101, wherein the protein of interest is an antibody or antigen binding fragment thereof. The antibody or antigen-binding fragment thereof,
(A) optionally said antibody or antigen binding fragment thereof comprises VH having the amino acid sequence of SEQ ID NO:5 or 6, and VL having the amino acid sequence of SEQ ID NO:7, specifically for TNF-like ligand 1A (TL1a) Join,
(B) optionally said antibody or antigen-binding fragment thereof comprises VH having the amino acid sequence of SEQ ID NO: 1 and VL having the amino acid sequence of SEQ ID NO: 2 and specifically binds to calcitonin gene-related peptide (CGRP) Or (c) optionally said antibody or antigen-binding fragment thereof comprises VH having the amino acid sequence of SEQ ID NO:3 and VL having the amino acid sequence of SEQ ID NO:4 and specifically binds to CD38,
103. The preparation according to claim 102. 101. A composition comprising a preparation of a protein of interest produced by the method of any one of claims 1-73 and 76-100 and a pharmaceutically acceptable excipient. 101. A composition comprising a virus inactivated preparation of a protein of interest produced by the method of any one of claims 1-73 and 76-100, and a pharmaceutically acceptable excipient. A composition comprising an aqueous carrier and a recombinantly expressed or hybridoma-expressed antibody or antigen-binding fragment thereof, comprising:
The composition is substantially free of viral particles,
The antibody or antigen-binding fragment thereof,
(A) one or more complementarity determining regions (CDRs) of VH specifically binding to TNF-like ligand 1A (TL1a) and having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and the amino acid sequence of SEQ ID NO: 7 Including one or more CDRs of a VL having
(B) One or more CDRs of VH having the amino acid sequence of SEQ ID NO: 1 and one or more CDRs of VL having the amino acid sequence of SEQ ID NO: 2 specifically bound to calcitonin gene-related peptide (CGRP) Or (c) one or more CDRs of VH specifically binding to CD38 and having the amino acid sequence of SEQ ID NO:3, and one or more CDRs of VL having the amino acid sequence of SEQ ID NO:4,
The composition. 107. The composition of claim 106, wherein the composition is virus particle free. The antibody or antigen-binding fragment thereof,
(A) comprising VH having the amino acid sequence of SEQ ID NO: 5 or 6, and VL having the amino acid sequence of SEQ ID NO: 7,
(B) VH having the amino acid sequence of SEQ ID NO: 1 and VL having the amino acid sequence of SEQ ID NO: 2, or (c) VH having the amino acid sequence of SEQ ID NO: 3 and VL having the amino acid sequence of SEQ ID NO: 4 including,
108. The composition of claim 106 or 107. A composition comprising an aqueous carrier and a recombinantly expressed or hybridoma-expressing antibody prepared by a method, comprising:
a) loading a mixture comprising said antibody and one or more contaminants, aggregates and/or viruses on a Protein A chromatography support,
b) washing the support with a Protein A chromatography wash containing one or more detergents to remove the one or more contaminating proteins, aggregates and/or viruses from the Protein A chromatography support. ,
c) eluting the antibody from the Protein A chromatography support,
d) loading the eluate from step c) on a cation exchange (CEX) chromatography support,
e) washing the CEX chromatography support with a CEX chromatography wash containing one or more detergents to remove the one or more contaminating proteins, aggregates and/or viruses from the support, And f) eluting the antibody from the CEX chromatography support to form a purified eluate of the antibody,
The composition prepared by the method, comprising: The antibody or antigen-binding fragment thereof,
(A) one or more complementarity determining regions (CDRs) of VH specifically binding to TNF-like ligand 1A (TL1a) and having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and the amino acid sequence of SEQ ID NO: 7 Including one or more CDRs of a VL having
(B) One or more CDRs of VH having the amino acid sequence of SEQ ID NO: 1 and one or more CDRs of VL having the amino acid sequence of SEQ ID NO: 2 specifically bound to calcitonin gene-related peptide (CGRP) Or (c) one or more CDRs of VH specifically binding to CD38 and having the amino acid sequence of SEQ ID NO:3, and one or more CDRs of VL having the amino acid sequence of SEQ ID NO:4,
110. The composition of claim 109. 112. The composition of claim 109 or 110, wherein the composition is substantially free of viral particles. 112. The composition of any one of claims 109-111, wherein the composition has not undergone anion exchange chromatography prior to the CEX chromatography. 113. The composition of any one of claims 109-112, wherein the composition has not undergone any ion exchange chromatography prior to the CEX chromatography.

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